C3b binding polypeptide

ABSTRACT

Polypeptides comprising a C3b binding region are disclosed, as well as nucleic acids and vectors encoding such polypeptides, and cells and compositions comprising such polypeptides. Also disclosed are uses and methods using the polypeptides for treating and preventing diseases and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase of International Application No.PCT/EP2019/050949 filed Jan. 15, 2019, which designated the U.S. andthat International Application was published under PCT Article 21(2) inEnglish. This application also includes a claim of priority under 35U.S.C. §119(a) and §365(b) to British patent application No. GB1800620.5 filed Jan. 15, 2018, the entirety of which is herebyincorporated by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a sequence listing named “SequenceListing” inASCII text file format, created on Jan. 20, 2021, being 148 kb in size,which is herein incorporated by reference as though fully disclosed.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology,immunology, and medicine. More specifically, the present inventionrelates to a polypeptide that binds to C3b.

BACKGROUND TO THE INVENTION

Age-related macular degeneration (AMD) is the leading cause of blindnessin the developed world: AMD is currently responsible for 8.7% of allglobal blind registrations and it is estimated that 196 million peoplewill be affected by 2020 (Wong et al. Lancet Glob Heal (2014)2:e106-16). AMD manifests as the progressive destruction of the macula,the central part of the retina at the back of the eye, leading to lossof central visual acuity. Early stages of the disease see morphologicalchanges in the macula, including first the loss of blood vessels in thechoriocapillaris (Whitmore et al., Prog Retin Eye Res (2015) 45:1-29)which are fenestrated blood vessels found in the choroid (a highlyvascularized layer that supplies oxygen and nutrition to the outerretina).

AMD is largely a genetic disease. Mutations in genes of the complementsystem, part of our immune system, are highly associated with increasedrisk of AMD. Indeed, it has become clear that over-activation ofcomplement is a main driver of disease pathogenesis and many examples ofcomplement over-activation can be seen in the choriocapillaris. The roleof complement in AMD is reviewed, for example, by Zipfel et al. Chapter2, in Lambris and Adamis (eds.), Inflammation and Retinal Disease:Complement Biology and Pathology, Advances in Experimental Medicine andBiology 703, Springer Science+Business Media, LLC (2010), which ishereby incorporated by reference in its entirety. Complement isactivated by the deposition onto a surface of protein C3b, apro-inflammatory breakdown product of immune system protein C3. C3bassociates with other proteins to form convertase enzyme complexes foractivating and amplifying complement responses, and initiates theamplification loop of the complement cascade, ultimately leading tocell/tissue destruction and a local inflammatory response (allcharacteristics of AMD).

The choriocapillaris is separated from the metabolically active retinalpigment epithelium (RPE) by Bruch’s membrane (BrM), a thin (2-4 µm),acellular, five-layered, extracellular matrix. The BrM serves two majorfunctions: the substratum of the RPE and a blood vessel wall. Thestructure and function of BrM is reviewed e.g. in Curcio and Johnson,Structure, Function and Pathology of Bruch’s Membrane, In: Ryan et al.(2013), Retina, Vol. 1, Part 2: Basic Science and Translation toTherapy. 5th ed. London: Elsevier, pp466-481, which is herebyincorporated by reference in its entirety.

C3b activation of complement on acellular structures, such as BrM andthe intercapillary septa (extracellular matrix filling the spacesbetween capillaries in the choriocapillaris), is regulated by proteins‘complement factor H’ (FH) and ‘complement factor I’ (FI). FI preventscomplement activation by cleaving C3b to an proteolytically-inactiveform, designated iC3b, which is unable to participate in convertaseassembly. However, iC3b is an opsonin and therefore a mediator ofleucocyte recruitment with a subsequent inflammatory response, whereasthe further breakdown products of C3b, iC3dg and C3d, are poor opsonins.In order to cleave C3b, FI requires the presence of a cofactor, examplesof which include the blood borne FH protein and the membrane-boundsurface co-factor ‘complement factor 1’ (CR1; CD35). CR1 is a membranereceptor expressed on a wide range of cells and is involved in immunecomplex clearance, phagocytosis, and complement regulation. As well asserving as a co-factor in the Fl-mediated cleavage of C3b, CR1 acts as aregulator of complement by accelerating the decay of C3 and C5convertases. CR1 structure and function is reviewed e.g. in Khera andDas, Mol Immunol (2009) 46(5): 761-772 and Jacquet et al., J Immunol(2013) 190(7): 3721-3731, both of which are hereby incorporated byreference in their entirety.

Hallmark lesions of early AMD, termed drusen, develop within BrMadjacent to the RPE layer (Bird et al, Surv Ophthalmol (1995)39(5):367-374). Drusen are formed from the accumulation of lipids andcellular debris, and include a swathe of complement activation products(Anderson et al., Prog Retin Eye Res (2009) 29:95-112; Whitcup et al.,Int J Inflam (2013) 1-10). The presence of drusen within BrM disruptsthe flow of nutrients from the choroid across this extracellular matrixto the RPE cells, which leads to cell dysfunction and eventual death. Asthe RPE cell monolayer supports the rod and cone cells of theneurosensory retina by providing nutrients and removing waste, theircell death causes dysfunction of photoreceptor cells and subsequent lossof visual acuity.

This represents one of the late stages of AMD, known as ‘dry’ AMD andalso as geographic atrophy, which represents around 90% of AMD cases. Inthe remaining percentage of cases of late-stage AMD, the presence ofdrusen promotes choroidal neovascularisation (CNV), where the increasedsynthesis of vascular endothelial growth factor (VEGF) by RPE cellspromotes new blood vessel growth from the choroid/choriocapillaris thatbreaks through BrM into the retina. These new blood vessels leak andeventually form scar tissue; this is referred to as ‘wet’ AMD.

‘Wet’ AMD, while only representing 10% of cases, is the most virulentform of late-stage AMD and has different disease characteristics to‘dry’ AMD. There are treatments for wet AMD, where for example theinjection of anti-VEGF agents into the vitreous of the eye can slow orreverse the growth of these blood vessels, although it cannot preventtheir formation in the first place. Geographic atrophy (‘dry’ AMD)remains untreatable.

SUMMARY OF THE INVENTION

The present invention provides polypeptides that bind to C3b which areuseful for treating or preventing complement-related diseases orconditions.

In one aspect, the present invention provides a polypeptide which iscapable of binding C3b, the polypeptide comprising an amino acidsequence having at least 85% identity to SEQ ID NO:4 and wherein thepolypeptide has a total length of 450 amino acids or fewer, for use in amethod of treating or preventing a complement-related disease orcondition.

Also provided is a nucleic acid encoding a polypeptide which is capableof binding C3b, the polypeptide comprising an amino acid sequence havingat least 85% identity to SEQ ID NO:4 and wherein the polypeptide has atotal length of 450 amino acids or fewer, for use in a method oftreating or preventing a complement-related disease or condition.

In another aspect, the present invention provides the use of apolypeptide which is capable of binding C3b, the polypeptide comprisingan amino acid sequence having at least 85% identity to SEQ ID NO:4 andwherein the polypeptide has a total length of 450 amino acids or fewer,in the manufacture of a medicament for treating or preventing acomplement-related disease or condition.

Also provided is the use of a nucleic acid encoding a polypeptide whichis capable of binding C3b, the polypeptide comprising an amino acidsequence having at least 85% identity to SEQ ID NO:4 and wherein thepolypeptide has a total length of 450 amino acids or fewer, in themanufacture of a medicament for treating or preventing acomplement-related disease or condition.

In another aspect, provided is a method of treating or preventing acomplement-related disease or condition, comprising administering to asubject a polypeptide which is capable of binding C3b, the polypeptidecomprising an amino acid sequence having at least 85% identity to SEQ IDNO:4 and wherein the polypeptide has a total length of 450 amino acidsor fewer.

In another aspect, provided is a method of treating or preventing acomplement-related disease or condition in a subject, comprisingmodifying at least one cell of the subject to express or comprise apolypeptide capable of binding C3b, the polypeptide comprising an aminoacid sequence having at least 85% identity to SEQ ID NO:4 and whereinthe polypeptide has a total length of 450 amino acids or fewer.

In some embodiments the complement-related disease or condition is anocular disease or condition.

In some embodiments the treatment or prevention of an ocular disease orcondition comprises modifying at least one ocular cell of a subject toexpress or comprise the polypeptide. In some embodiments the treatmentor prevention of an ocular disease or condition comprises modifying atleast one ocular cell of a subject to express or comprise a nucleic acidencoding the polypeptide. In some embodiments the treatment orprevention of an ocular disease or condition comprises administering avector comprising a nucleic acid encoding the polypeptide to at leastone ocular cell of a subject. In some embodiments the at least oneocular cell is a retinal pigment epithelial (RPE) cell.

In some embodiments the disease or condition is a disease or conditionin which C3b or a C3b-containing complex, an activity/responseassociated with C3b or a C3b-containing complex, or a product of anactivity/response associated with C3b or a C3b-containing complex ispathologically implicated.

In some embodiments the disease or condition is macular degeneration. Insome embodiments the disease or condition is selected from one or moreof: age-related macular degeneration (AMD), early AMD, intermediate AMD,late AMD, geographic atrophy (‘dry’ AMD), ‘wet’ (neovascular) AMD,choroidal neovascularisation (CNV), glaucoma, autoimmune uveitis,diabetic retinopathy, and early-onset macular degeneration (EOMD).

In some embodiments the polypeptide comprises an amino acid sequencehaving at least 95% identity to SEQ ID NO:4. In some embodiments, X₁ isA orT, X₂ is P or L, and/or X₃ is G or R of SEQ ID NO:4.

In some embodiments the polypeptide has a total length of 50 to 250amino acids. In some embodiments the polypeptide comprises, or consistsof, SEQ ID NO:2 or SEQ ID NO:3.

In some embodiments the polypeptide comprises, or consists of, SEQ IDNO:13.

In some embodiments the polypeptide is capable of acting as a co-factorfor Complement Factor l. In some embodiments the polypeptide is capableof diffusing across Bruch’s membrane (BrM). In some embodiments thepolypeptide binds to C3b in the region bound by a co-factor forComplement Factor I. In some embodiments the polypeptide binds to C3b inthe region bound by Complement Receptor 1 (CR1).

In some embodiments the polypeptide comprises a secretory pathwaysequence. In some embodiments the secretory pathway sequence comprises,or consists of, SEQ ID NO:7. In some embodiments the polypeptidecomprises, or consists of, SEQ ID NO:47, 49, or 51. In some embodimentsthe polypeptide comprises a cleavage site for removing the secretorypathway sequence.

In one aspect, the present invention provides a polypeptide having atleast 80% sequence identity to SEQ ID NO:4, wherein the polypeptide hasa length of 700 amino acids or fewer.

In some embodiments, the polypeptide has a length of 50 to 700 aminoacids. In some embodiments, the polypeptide has at least 80% sequenceidentity to SEQ ID NO:4 wherein X₁ is A or T, X₂ is P or L, and/or X₃ isG or R.

Also provided is a polypeptide which is capable of binding to C3b, thepolypeptide comprising an amino acid sequence having at least 85%sequence identity to SEQ ID NO:4 and wherein the polypeptide has a totallength of 450 amino acids or fewer.

In some embodiments the polypeptide comprises an amino acid sequencehaving at least 95% identity to SEQ ID NO:4. In some embodiments X₁ is Aor T, X₂ is P or L, and/or X₃ is G or R.

In some embodiments the polypeptide has a total length of 50 to 250amino acids.

In some embodiments, the polypeptide comprises, or consists of, an aminoacid sequence according to SEQ ID NO:2. In some embodiments, thepolypeptide comprises, or consists of, an amino acid sequence accordingto SEQ ID NO:3. In some embodiments, the polypeptide comprises, orconsists of, an amino acid sequence according to SEQ ID NO:13.

In some embodiments, the polypeptide is capable of binding to C3b. Insome embodiments, the polypeptide binds to C3b in the region bound by aco-factor for Complement Factor I. In some embodiments, the polypeptidebinds to C3b in the region bound by Complement Receptor 1 (CR1).

In some embodiments, the polypeptide acts as a co-factor for ComplementFactor I.

In some embodiments, the polypeptide is capable of diffusing acrossBruch’s membrane (BrM). In some embodiments, the polypeptide is notglycosylated or is partially glycosylated. In some embodiments, thepolypeptide comprises at least one amino acid substitution, e.g. one,two, three or four substitutions, at position 509, 578, 959 and/or 1028(numbered according to Uniprot: P17927 (SEQ ID NO:1)). In someembodiments, the at least one amino acid substitution is one or more ofN509Q, N578Q, N959Q and/or N1028Q (numbered according to Uniprot: P17927(SEQ ID NO:1)). In some embodiments, the polypeptide comprises, orconsists of, SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:15.

In some embodiments, the polypeptide additionally comprises a secretorypathway sequence. In some embodiments, the secretory pathway sequencecomprises, or consists of, SEQ ID NO:7. In some embodiments, thepolypeptide additionally comprises a cleavage site for removing thesecretory pathway sequence. In some embodiments the polypeptidecomprises, or consists of, SEQ ID NO:47, 49 or 51.

In another aspect, the present invention provides a nucleic acidencoding a polypeptide according to the present invention.

In another aspect, the present invention provides a vector comprising anucleic acid of the present invention.

In another aspect, the present invention provides a cell comprising apolypeptide, nucleic acid, or vector according to the present invention.

In another aspect, the present invention provides a method for producinga polypeptide, comprising introducing into a cell a nucleic acid or avector according to the present invention, and culturing the cell underconditions suitable for expression of the polypeptide.

In another aspect, the present invention provides a cell, which isobtained or obtainable by the method for producing a polypeptideaccording to the present invention.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a polypeptide, nucleic acid, vector or cellaccording to the present invention. In some embodiments, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier, adjuvant, excipient, or diluent.

In another aspect, the present invention provides a polypeptide, nucleicacid, vector, cell or pharmaceutical composition according to thepresent invention, for use in a method of treating or preventing adisease or condition.

In another aspect, the present invention provides the use of apolypeptide, nucleic acid, vector, cell or pharmaceutical compositionaccording to the present invention, in the manufacture of a medicamentfor treating or preventing a disease or condition.

In another aspect, the present invention provides a method of treatingor preventing a disease or condition, comprising administering to asubject a polypeptide, nucleic acid, vector, cell or pharmaceuticalcomposition according to the present invention.

In another aspect, the present invention provides a method of treatingor preventing a disease or condition in a subject, comprising modifyingat least one cell of the subject to express or comprise a nucleic acid,vector or polypeptide according to the present invention.

In some embodiments in accordance with various aspects of the presentinvention, the disease or condition is a disease or condition in whichC3b or a C3b-containing complex, an activity/response associated withC3b or a C3b-containing complex, or a product of an activity/responseassociated with C3b or a C3b-containing complex is pathologicallyimplicated. In some embodiments, the disease or condition is maculardegeneration. In some embodiments, the disease or condition isage-related macular degeneration (AMD). In some embodiments, the methodfor treating or preventing a disease or condition comprises modifying atleast one retinal pigment epithelial (RPE) cell of the subject toexpress or comprise a nucleic acid, vector, or polypeptide according tothe present invention.

In another aspect, the present invention provides a kit of partscomprising a predetermined quantity of a polypeptide, nucleic acid,vector, cell, or pharmaceutical composition according to the presentinvention.

DESCRIPTION

Complement-based therapies for AMD have thus far concentrated uponinjecting complement regulating antibodies into the eye. Such therapieshave provided little to no therapeutic benefit as these proteins cannotreach the target area, i.e. the BrM and its underlying vasculature, thechoriocapillaris at all, or in effective concentrations.

Complement Factor I (FI)-mediated regulation of complement, i.e. thecleavage of C3b to iC3b (proteolytically-inactive C3b), requirescofactors such as membrane-anchored CR1. However, the present inventorshave discovered that it is not necessary to provide full-lengthmembrane-bound CR1, nor even a soluble version of CR1 merely lacking thetransmembrane domain, for successful cofactor activity. Instead, theinventors have discovered that short CR1 fragments comprising the CR1C3b-binding domains are sufficient to enable efficient FI-mediated C3bcleavage.

Thus, the present invention relates to soluble, truncated polypeptidesderived from the FI cofactor CR1. The polypeptides comprise domains thatare capable of binding to C3b, such that they can act as essential Flcofactors for the regulation of complement activation. A key advantageof the soluble, truncated polypeptides is their ability to pass throughBrM, and thus they are able to reach all regions associated with AMD,i.e. the RPE/BrM interface, BrM and the choroid, including theintercapillary septa (the extracellular matrix between the blood vesselsof the choriocapillaris). The present invention also providesnon-glycosylated polypeptides derived from CR1, which may aidpolypeptide passage through BrM. The polypeptides are expressed andsecreted easily, enabling in situ expression by cells local to theaffected sites and targeting of the polypeptides to areas affected bycomplement over-activation. In situ expression of the polypeptides maybe achieved using gene therapy techniques. In situ expression providestargeted therapy to areas of need without disrupting functioningcomplement regulation elsewhere in the body.

The present invention enables supplementation of a deterioratingcomplement regulation system without replacing the endogenous complementregulation currently in place, or interfering in the rest of thecomplement cascade.

Polypeptides

A polypeptide according to the present invention may comprise, orconsist of, one or more C3b binding regions.

A polypeptide according to the present invention has at least 80%sequence identity to SEQ ID NO:4, wherein the polypeptide has a lengthof 700 amino acids or fewer. In some embodiments, the polypeptidecomprises, or consists of, an amino acid sequence having at least 80%sequence identity to SEQ ID NO:4, wherein the polypeptide has a lengthof 700 amino acids or fewer.

A polypeptide according to the present invention may comprise, orconsist of, an amino acid sequence of 700 amino acids or fewer having atleast 80% sequence identity to SEQ ID NO:4.

In some embodiments, the polypeptide comprises, or consists of, an aminoacid sequence of 650, 600, 550, 500, 450, 400, 350, 300, 250, or 200amino acids or fewer. In some embodiments, the polypeptide comprises, orconsists of, an amino acid sequence having 1 to 200 amino acids, 1 to250 amino acids, 1 to 300 amino acids, 1 to 350 amino acids, 1 to 400amino acids, 1 to 450 amino acids, 1 to 500 amino acids, 1 to 550 aminoacids, 1 to 600 amino acids, 1 to 650 amino acids, or 1 to 700 aminoacids. In some embodiments, the polypeptide has a length of 50 to 700amino acids. In some embodiments, the polypeptide has a length of 100 to650 amino acids. In some embodiments, the polypeptide has a length of100 to 550 amino acids. In some embodiments, the polypeptide has alength of 150 to 450 amino acids. In some embodiments, the polypeptidehas a length of 400 to 700 amino acids. In some embodiments, thepolypeptide has a length of 700 to 1000 or greater than 1000 aminoacids.

“Length” as used herein refers to the total length of the polypeptide;that is, “length” refers to the measurement or extent of the entirepolypeptide from end to end, i.e. from the N-terminus to the C-terminus.“Length” as used herein is measured by the number of amino acid residueswithin the polypeptide.

In some embodiments the polypeptide is adetached/discrete/separate/individual molecule. In some embodiments, thepolypeptide is a single contiguous amino acid sequence that isunconnected, i.e. not joined, fused or attached, to another amino acidsequence. In some embodiments the polypeptide is not attached by anamino acid linker or a non-amino acid linker to another polypeptide oramino acid sequence. In some embodiments the polypeptide is not asection, part or region of a longer amino acid sequence, i.e. it is notpart of an amino acid sequence that exceeds the maximum, specified,polypeptide length. In some embodiments the polypeptide is not part of,or does not form a section of, a fusion protein. In some embodiments thepolypeptide may comprise a sequence provided herein and one or moreadditional amino acids, as long as the maximum length of the polypeptideis not exceeded. The short length of the polypeptides described hereinenables the polypeptides to pass through the BrM and reach sites ofcomplement activation.

In some embodiments the polypeptide has a total length of 700, 650, 600,550, 500, 450, 400, 350, 300, 250, or 200 amino acids or fewer. In someembodiments the polypeptide has a total length of 450, 440, 430, 420,410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280,270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140,130, 120, 110, 100, 90, 80, or 70 amino acids or fewer. In someembodiments the polypeptide has a total length of 1 to 70 amino acids, 1to 80 amino acids, 1 to 90 amino acids, 1 to 100 amino acids, 1 to 110amino acids, 1 to 120 amino acids, 1 to 130 amino acids, 1 to 140 aminoacids, 1 to 150 amino acids, 1 to 160 amino acids, 1 to 170 amino acids,1 to 180 amino acids, 1 to 190 amino acids, 1 to 200 amino acids, 1 to210 amino acids, 1 to 220 amino acids, 1 to 230 amino acids, 1 to 240amino acids, 1 to 250 amino acids, 1 to 260 amino acids, 1 to 270 aminoacids, 1 to 280 amino acids, 1 to 290 amino acids, 1 to 300 amino acids,1 to 310 amino acids, 1 to 320 amino acids, 1 to 330 amino acids, 1 to340 amino acids, 1 to 350 amino acids, 1 to 360 amino acids, 1 to 370amino acids, 1 to 380 amino acids, 1 to 390 amino acids, 1 to 400 aminoacids, 1 to 410 amino acids, 1 to 420 amino acids, 1 to 430 amino acids,1 to 440 amino acids, or 1 to 450 amino acids. In some embodiments thepolypeptide has a total length of 50 to 450 amino acids, 50 to 400 aminoacids, 50 to 350 amino acids, 50 to 300 amino acids, 50 to 250 aminoacids, 50 to 200 amino acids, 100 to 250 amino acids, 100 to 200 aminoacids, 150 to 250 amino acids, or 150 to 200 amino acids. In someembodiments the polypeptide has a total length of one of 61, 72, 194,212, 231, 388, 406, or 644 amino acids.

In some embodiments a polypeptide of the present invention has a maximummolecular weight of 80 kDa, whether the polypeptide iscovalently/non-covalently bonded to a larger complex, part of a largercomplex, or is not part of a larger complex. In some embodiments apolypeptide of the present invention has a molecular weight of 75 kDa orless, 70 kDa or less, 65 kDa or less, 60 kDa or less, 55 kDa or less, 50kDa or less, 45 kDa or less, 40 kDa or less, 35 kDa or less, 30 kDa orless, 29 kDa or less, 28 kDa or less, 27 kDa or less, 26 kDa or less, 25kDa or less, 24 kDa or less, 23 kDa or less, 22 kDa or less, 21 kDa orless, 20 kDa or less, 19 kDa or less, 18 kDa or less, 17 kDa or less, 16kDa or less, 15 kDa or less, 14 kDa or less, 13 kDa or less, 12 kDa orless, 11 kDa or less, or 10 kDa or less. In some embodiments thepolypeptide has a maximum molecular weight of 50 kDa, i.e. 50 kDa orless. In some embodiments the polypeptide has a maximum molecular weightof 26 kDa, i.e. 26 kDa or less. In some embodiments the polypeptide hasa maximum molecular weight of 24 kDa, i.e. 24 kDa or less. In someembodiments the polypeptide has a maximum molecular weight of 22 kDa,i.e. 22 kDa or less. In some embodiments the polypeptide has a maximummolecular weight of 20 kDa, i.e. 20 kDa or less.

In some embodiments, a polypeptide of the present invention comprises,or consists of, an amino acid sequence having at least 70%, 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4. Insome embodiments, X₁ is A or T, X₂ is P or L, and/or X₃ is G or R. Insome embodiments, X₁ is A, X₂ is P, and/or X₃ is G. In some embodiments,X₁ is A, X₂ is L, and/or X₃ is R. In some embodiments, X₁ is A, X₂ is P,and/or X₃ is R. In some embodiments, X₁ is A, X₂ is L, and/or X₃ is G.In some embodiments, X₁ is T, X₂ is L, and/or X₃ is R. In someembodiments, X₁ is T, X₂ is P, and/or X₃ is G. In some embodiments, X₁is T, X₂ is L, and/or X₃ is G. In some embodiments, X₁ is T, X₂ is P,and/or X3 is R.

In some embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO:4, wherein the polypeptide has a lengthprovided herein. For example, in some embodiments the polypeptidecomprises, or consists of, an amino acid sequence having 85% sequenceidentity to SEQ ID NO:4, wherein the polypeptide has a total length of450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320,310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180,170, or 160 amino acids or fewer; in some embodiments the polypeptidecomprises, or consists of, an amino acid sequence having 90% sequenceidentity to SEQ ID NO:4, wherein the polypeptide has a total length of450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320,310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, or 180amino acids or fewer; in some embodiments the polypeptide comprises, orconsists of, an amino acid sequence having 95% sequence identity to SEQID NO:4, wherein the polypeptide has a total length of 450, 440, 430,420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290,280, 270, 260, 250, 240, 230, 220, 210, 200, 190, or 180 amino acids orfewer; in some embodiments the polypeptide comprises, or consists of, anamino acid sequence having 98% sequence identity to SEQ ID NO:4, whereinthe polypeptide has a total length of 450, 440, 430, 420, 410, 400, 390,380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250,240, 230, 220, 210, or 200 amino acids or fewer.

In some embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 85% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 450 amino acids or fewer.In some embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 85% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 50 to 450 amino acids. Insome embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 85% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 250 amino acids or fewer.In some embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 85% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 50 to 250 amino acids. Insome embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 95% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 450 amino acids or fewer.In some embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 95% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 50 to 450 amino acids. Insome embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 95% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 250 amino acids or fewer.In some embodiments the polypeptide comprises, or consists of, an aminoacid sequence having at least 95% sequence identity to SEQ ID NO:4,wherein the polypeptide has a total length of 50 to 250 amino acids.

Human CR1 (UniProt: P17927 (Entry version 181 (25 Oct. 2017), Sequenceversion 3 (02 Mar. 2010)); SEQ ID NO:1) has a 2,039 amino acid sequence(including an N-terminal, 41 amino acid signal peptide), and comprises30 complement control protein (CCP) domains (also known as sushi domainsor short consensus repeats (SCRs)), with the N-terminal 28 CCPsorganised into four long homologous repeat (LHR) domains each comprising7 CCPs: LHR-A, LHR-B, LHR-C and LHR-D. The C3b binding region of CR1 isfound in CCPs 8-10 in LHR-B (UniProt: P17927 positions 491 to 684; SEQID NO:2), and CCPs 15-17 in LHR-C (UniProt: P17927 positions 941 to1134; SEQ ID NO:3). CCPs 8-10 and 15-17 differ in sequence by threeamino acid residues, as shown in consensus sequence SEQ ID NO:4.

A polypeptide according to the present invention may comprise, orconsist of, an amino acid sequence corresponding to CCPs 8-10 (SEQ IDNO:2) and/or CCPs 15-17 (SEQ ID NO:3). In some embodiments, apolypeptide of the present invention comprises, or consists of, an aminoacid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO:2 and/or SEQ ID NO:3. Suchpolypeptides may have any length provided herein.

A polypeptide according to the present invention may comprise, orconsist of, an amino acid sequence corresponding to CCPs 8-10 and 15-17.The polypeptide may comprise or consist of CCPs 8-10 and 15-17 in theirnative CR1 sequence (SEQ ID NO:30). The polypeptide may comprise orconsist of CCPs 8-10 joined to CCPs 15-17. This may be in a contiguoussequence (SEQ ID NO:13), or achieved by a linker between CCPs 8-10 and15-17 (e.g. SEQ ID NO:14). In some embodiments, a polypeptide of thepresent invention comprises, or consists of, an amino acid sequencehaving at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO:13, SEQ ID NO:14 and/or SEQ ID NO:30. Suchpolypeptides may have any length provided herein.

A polypeptide according to the present invention may comprise, orconsist of, an amino acid sequence corresponding to one or more ofsequence ‘A’ (SEQ ID NO:8), sequence ‘B’ (SEQ ID NO:16) and/or ‘SequenceC’ (SEQ ID NO:17). In some embodiments, the polypeptide consists of asequence selected from sequence ‘A’ (SEQ ID NO:8), sequence ‘B’ (SEQ IDNO:16) and ‘Sequence C’ (SEQ ID NO:17). In some embodiments, apolypeptide of the present invention comprises, or consists of, an aminoacid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to one or more of sequences ‘A’ (SEQ ID NO:8),‘B’ (SEQ ID NO:16) and/or ‘C’ (SEQ ID NO:17).

In some embodiments, where a polypeptide according to the presentinvention comprises, or consists of, sequence ‘B’ (SEQ ID NO:16) or anamino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to sequence ‘B’ (SEQ ID NO:16), X₁ is Aor T. In some embodiments, X₁ is A. In some embodiments, X₁ is T.

In some embodiments, where a polypeptide according to the presentinvention comprises, or consists of, sequence ‘C’ (SEQ ID NO:17) or anamino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to sequence ‘C’ (SEQ ID NO:17), X₂ is Por L and/or X₃ is G or R. In some embodiments, X₂ is P and/or X₃ is G.In some embodiments, X₂ is P and/or X₃ is R. In some embodiments, X₂ isL and/or X₃ is G. In some embodiments, X₂ is L and/or X₃ is R.

In some embodiments sequence ‘B’ corresponds to SEQ ID NO:9 or SEQ IDNO:11. In some embodiments sequence ‘C’ corresponds to SEQ ID NO:10 orSEQ ID NO:12.

The present invention includes polypeptides comprising sequences ‘A’,‘B’, and/or ‘C’ as described herein, and combinations thereof, includingat least the following combinations (organised from N-terminus toC-terminus):

-   A + B-   B + C-   A + C-   C + A-   A + B + C-   B + C + A-   C + A + B-   A + B + C + A-   B + C + A + B-   C + A + B + C-   A + B + C + A + B-   B + C + A + B + C-   A + B + C + A + B + C-   A + B + C + A + B + C + Y (where Y = one or more of A, B and/or C).

In some embodiments, the polypeptide comprises, or consists of, an aminoacid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to any combination of CCP domains or anycombination of sequences ‘A’, ‘B’ and/or ‘C’ described herein.

In some embodiments, the combination of CCP domains is a combinationfound in native CR1. In some embodiments, the combination of CCP domainsis not a combination found in native CR1.

In some embodiments, the polypeptide comprises, or consists of, an aminoacid sequence having multiple copies of sequence ‘A’, multiple copies ofsequence ‘B’, and/or multiple copies of sequence ‘C’. In someembodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8 or morecopies of one or more of sequences ‘A’, ‘B’ and/or ‘C’. In someembodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8 or morecopies of sequence ‘A’; 1, 2, 3, 4, 5, 6, 7, 8 or more copies ofsequence ‘B’; and/or 1, 2, 3, 4, 5, 6, 7, 8 or more copies of sequence‘C’. In some embodiments, the polypeptide comprises 9, 10, or morecopies of one or more of sequences ‘A’, ‘B’ and/or ‘C’.

In some embodiments, a polypeptide according to the present inventionlacks substantial sequence identity to one or more of amino acidsequences 1-490, 685-940 and/or 1135-2039 of human CR1 (SEQ ID NO:1). Apolypeptide that lacks substantial sequence identity as described hereinmay have less than 80%, less than 75%, less than 70%, less than 65%,less than 60%, less than 55%, less than 50%, less than 45%, less than40%, less than 35%, less than 30%, less than 25%, less than 20%, lessthan 15%, less than 10%, or less than 5% sequence identity to one ormore of amino acid sequences 1-490, 685-940 and/or 1135-2039 of humanCR1 (SEQ ID NO:1). In some embodiments a polypeptide according to thepresent invention lacks amino acid sequence having substantial sequenceidentity to CR1 long homologous repeat (LHR) domains LHR-A and/or LHR-D.In some embodiments a polypeptide according to the present inventionlacks amino acid sequence having substantial sequence identity to CR1CCP domains 1-7, 11-14 and/or 18-30. Amino acid residues of SEQ ID NO:1are numbered according to Uniprot P17927; Entry version 181 (25 Oct.2017), Sequence version 3 (02 Mar. 2010).

A polypeptide according to the present invention, and/or describedherein, may be isolated and/or substantially purified.

Further Features of the Polypeptide

Polypeptides according to the present invention may comprisemodifications and/or additional amino acid sequences. The modificationsand/or additional amino acid sequences may be included in the lengthlimitation of a polypeptide provided herein such that the lengthlimitation of that polypeptide is not exceeded.

In some embodiments, an additional amino acid sequence comprises, orconsists of, no more than 25, 50, 100, 150, or 200 amino acids, i.e. anadditional amino acid sequence comprises, or consists of, 1-25, 1-50,1-100, 1-150, or 1-200 amino acids. In some embodiments, an additionalamino acid sequence comprises more than 200 amino acids. In someembodiments, an additional amino acid sequence comprises no more than100 amino acids at the C-terminus of a polypeptide according to thepresent invention, and/or no more than 100 amino acids at the N-terminusof a polypeptide according to the present invention.

In some embodiments, an additional amino acid sequence results in apolypeptide longer than 700 amino acids. In some embodiments, apolypeptide according to the present invention comprises, or consistsof, 700 or more amino acids. For example, the polypeptide may comprise,or consist of, 700-750, 750-800, 800-850, 850-900, 900-950, 950-100, ormore than 1000 amino acids.

In some embodiments, an additional amino acid sequence described hereinlacks substantial sequence identity to one or more of amino acidsequences 1-490, 685-940 and/or 1135-2039 of human CR1 (SEQ ID NO:1,numbered according to Uniprot P17927; Entry version 181 (25 Oct. 2017),Sequence version 3 (02 Mar. 2010)). In some embodiments, the additionalamino acid sequence lacks substantial sequence identity to CR1 CCPdomains 1-7, 11-14 and/or 18-30. In some embodiments, the additionalamino acid sequence has less than 80%, less than 75%, less than 70%,less than 65%, less than 60%, less than 55%, less than 50%, less than45%, less than 40%, less than 35%, less than 30%, less than 25%, lessthan 20%, less than 15%, less than 10%, or less than 5% sequenceidentity to one or more of amino acid sequences 1-490, 685-940 and/or1135-2039 of human CR1 (SEQ ID NO:1). In some embodiments the additionalamino acid sequence lacks substantial sequence identity to CR1 longhomologous repeat (LHR) domains LHR-A and/or LHR-D.

In some embodiments, a polypeptide may lack amino acid sequence havingsubstantial sequence identity to a region of a co-factor for ComplementFactor I (e.g. CR1) other than in the C3b binding region. For example,the polypeptide may lack amino acid sequence having substantial sequenceidentity to CR1 other than in CR1 CCP domains 8-10 and/or 15-17(residues 491 to 684 and/or 941 to 1134, respectively, of SEQ ID NO:1).In some embodiments, the polypeptide may lack amino acid sequence havingsubstantial sequence identity to CR1 CCP domains 1-7, 11-14 and/or18-30. A polypeptide lacking amino acid sequence having substantialsequence identity as described herein may have less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, lessthan 50%, less than 45%, less than 40%, less than 35%, less than 30%,less than 25%, less than 20%, less than 15%, less than 10%, or less than5% sequence identity to one or more of amino acid sequences 1-490,685-940 and/or 1135-2039 of human CR1 (SEQ ID NO:1).

In some embodiments, a polypeptide according to the present inventionmay comprise a secretory pathway sequence. As used herein, a secretorypathway sequence is an amino acid sequence which directs secretion ofpolypeptide. The secretory pathway sequence may be cleaved from themature protein once export of the polypeptide chain across the roughendoplasmic reticulum is initiated. Polypeptides secreted by mammaliancells generally have a signal peptide fused to the N-terminus of thepolypeptide, which is cleaved from the translated polypeptide to producea “mature” form of the polypeptide.

In some embodiments, the secretory pathway sequence may comprise orconsist of a leader sequence (also known as a signal peptide or signalsequence). Leader sequences normally consist of a sequence of 5-30hydrophobic amino acids, which form a single alpha helix. Secretedproteins and proteins expressed at the cell surface often compriseleader sequences. The leader sequence may be present in thenewly-translated polypeptide (e.g. prior to processing to remove theleader sequence). Leader sequences are known for many proteins, and arerecorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL,Protein Information Resource, Protein Data Bank, Ensembl, and InterPro,and/or can be identified/predicted e.g. using amino acid sequenceanalysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

In some embodiments, the secretory pathway sequence is derived fromComplement Factor H (FH). In some embodiments, the secretory pathwaysequence comprises or consists of SEQ ID NO:7. In some embodiments, thesecretory pathway sequence of the polypeptide of the present inventioncomprises, or consists of, an amino acid sequence having at least 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:7.

In some embodiments a polypeptide according to the present inventioncomprises, or consists of, an amino acid sequence corresponding to SEQID NO:47, 49, and/or 51. In some embodiments the polypeptide comprises,or consists of, an amino acid sequence having at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:47, 49 and/or 51.Such polypeptides may have any length provided herein.

In some embodiments, a polypeptide according to the present inventionmay additionally comprise a cleavage site for removing the secretorypathway sequence from the polypeptide. In some embodiments, the cleavagesite for removing the secretory pathway sequence from the polypeptide isa cleavage site for an endoprotease. In some embodiments, the cleavagesite is for an endoprotease expressed by the cell in which thepolypeptide is expressed. In some embodiments, the cleavage site is asignal peptidase cleavage site. In some embodiments, the cleavage siteis a protease cleavage site, e.g. a cleavage site for an endoproteaseexpressed by cells expressing the polypeptide. In some embodiments, thecleavage site is a cleavage site for an endoprotease expressed by RPEcells.

A polypeptide according to the present invention may comprise one ormore linker sequences between amino acid sequences. A linker sequencemay be provided between any two or more of sequences ‘A’, ‘B’ and/or‘C’. In some embodiments, a polypeptide according to the presentinvention comprises, or consists of, an amino acid sequence A + B + C-[LINKER]-A + B + C. In some embodiments, a polypeptide comprises, orconsists of, SEQ ID NO:14.

Linker sequences are known to the skilled person, and are described, forexample in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369,which is hereby incorporated by reference in its entirety. In someembodiments, a linker sequence may be a flexible linker sequence.Flexible linker sequences allow for relative movement of the amino acidsequences which are linked by the linker sequence. Flexible linkers areknown to the skilled person, and several are identified in Chen et al.,Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequencesoften comprise high proportions of glycine and/or serine residues.

In some embodiments, the linker sequence comprises at least one glycineresidue and/or at least one serine residue. In some embodiments thelinker sequence consists of glycine and serine residues. In someembodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5,1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 amino acids.

In some embodiments, a polypeptide according to the present inventioncomprises a non-amino acid linker. In some embodiments, a polypeptideaccording to the present invention may comprise two or more polypeptideslinked by conjugation, e.g. by nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides, active esters),electrophilic substitutions (e.g., enamine reactions) and additions tocarbon-carbon and carbon-heteroatom multiple bonds (e.g., Michaelreaction, Diels-Alder addition). These and other useful reactions arediscussed in, for example, March, Advanced Organic Chemistry, 3rd Ed.,John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques,Academic Press, San Diego, 1996; and Feeney et al., Modification ofProteins; Advances in Chemistry Series, Vol. 198, American ChemicalSociety, Washington, D.C., 1982.

In some embodiments, a polypeptide according to the present inventioncomprises a cleavable linker.

It may be desirable for a polypeptide according to the present inventionto lack certain properties of CR1. For example, it may be desirable forthe polypeptide to lack regions that would otherwise inhibit diffusionthrough Bruch’s membrane (BrM) or that would interfere with the actionof native co-factor family proteins.

A polypeptide according to the present invention lacks the CR1transmembrane domain (SEQ ID NO:32). A polypeptide according to thepresent invention may lack the CR1 cytoplasmic tail (SEQ ID NO:33). In apreferred embodiment, a polypeptide according to the present inventionis soluble.

A polypeptide according to the present invention may lack regions whichcould otherwise be exploited by pathogenic bacteria to subvert the hostimmune system. Bacteria have developed molecules on their surface thatcan bind and recruit soluble complement factor H from the blood. Thisenables the bacteria to effectively coat themselves in a complementregulator and evade a host immune response. Polypeptides according tothe present invention may lack bacterial binding sites such that theycannot be used by invading pathogens to evade an immune response.

In some embodiments a polypeptide according to the present inventioncomprises one or more sites for glycosylation. In some embodiments apolypeptide according to the present invention is glycosylated.

In some embodiments, a polypeptide according to the present invention isnot glycosylated. In some embodiments, a polypeptide according to thepresent invention lacks one or more sites for glycosylation. In someembodiments, the polypeptide of the present invention lacks one or moresites for N-linked glycosylation. In some embodiments, a polypeptideaccording to the present invention lacks N-linked glycans. In someembodiments, a polypeptide according to the present invention isexpressed and/or secreted by cells that are unable to glycosylate orfully glycosylate polypeptides. For example, cells may lack functionalglycosyl transferase enzymes. In some embodiments, the polypeptide isaglycosyl (i.e. is not glycosylated). In some embodiments, thepolypeptide has been deglycosylated, e.g. by treatment with aglycosidase (e.g. Peptide N-Glycosidase). Deglycosylation is preferablynon-denaturing. In some embodiments a polypeptide according to thepresent invention is partially glycosylated, non-glycosylated orde-glycosylated.

In some embodiments, a polypeptide according to the present inventionlacks sequence conforming to the consensus sequence of SEQ ID NO:27. Insome embodiments, the polypeptide according to the present inventioncomprises one or more sequences conforming to the consensus sequence ofSEQ ID NO:27 that have been mutated to remove sites for N-glycosylation.In some embodiments, the Asn (N) residue in one or more consensussequences according to SEQ ID NO:27 is substituted with another aminoacid residue, e.g. a residue selected from: Ala (A), Cys (C), Asp (D),Glu (E), Phe (F), Gly (G), His (H), Iie (I), Lys (K), Leu (L), Met (M),Pro (P), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), Trp (W) or Tyr(Y). In some embodiments, the Asn (N) residue in one or more consensussequences according to SEQ ID NO:27 is substituted with a Gln (Q)residue. In some embodiments, residue X₂ of SEQ ID NO:27 is, or ismutated to be, an amino acid that is not Ser (S) or Thr (T).

In some embodiments, a polypeptide comprising, or consisting of, anamino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 comprisesone or more amino acid substitutions at position 509 and/or position 578(numbered according to Uniprot: P17927). In some embodiments, the one ormore amino acid substitutions are selected from N509Q and/or N578Q. Insome embodiments, a polypeptide comprising, or consisting of, an aminoacid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO:3 comprises one or more amino acidsubstitutions at position 959 and/or position 1028 (numbered accordingto Uniprot: P17927). In some embodiments, the one or more amino acidsubstitutions are selected from N959Q and/or N1028Q. In someembodiments, a polypeptide comprising, or consisting of, an amino acidsequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO:13 comprises one or more amino acidsubstitutions at position 509, 578, 959 and/or position 1028 (numberedaccording to Uniprot: P17927). In some embodiments, the one or moreamino acid substitutions are selected from N509Q, N578Q, N959Q and/orN1028Q. Such polypeptides may have any length provided herein.

In some embodiments, the polypeptide comprises, or consists, of an aminoacid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:15, and/orSEQ ID NO:31. Such polypeptides may have any length provided herein.

In some embodiments a polypeptide according to the present inventioncomprises a secretory pathway sequence and one or more sequencesconforming to the consensus sequence of SEQ ID NO:27 that have beenmutated to remove sites for N-glycosylation. In some embodiments apolypeptide according to the present invention comprises, or consistsof, an amino acid sequence corresponding to SEQ ID NO:48, 50, 52, 53,and/or 54. In some embodiments the polypeptide comprises, or consists,of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to SEQ ID NO:48, 50, 52, 53, and/or54. Such polypeptides may have any length provided herein.

In some embodiments, a polypeptide according to the present inventionmay comprise amino acid sequence(s) to facilitate expression, folding,trafficking, processing, purification or detection of the polypeptide.For example, the polypeptide may comprise a sequence encoding a proteintag, e.g. a His, (e.g. 6XHis), FLAG, Myc, GST, MBP, HA, E, or Biotintag, optionally: at the N— or C— terminus of the polypeptide; in alinker; or at the N— or C— terminus of a linker. In some embodiments thepolypeptide comprises a detectable moiety, e.g. a fluorescent,luminescent, immuno-detectable, radio, chemical, nucleic acid orenzymatic label. In some embodiments, the detectable moiety facilitatesdetection of the polypeptide in a sample obtained from a subject, e.g.following administration to the subject of the polypeptide, nucleicacid, vector, cell or pharmaceutical composition according to thepresent invention. The sample may be any biological sample obtained froma subject. In some embodiments the sample is a liquid biopsy, such asocular fluid (tear fluid, aqueous humour, or vitreous), blood, plasma,etc. In some embodiments the sample is a cytological sample or a tissuesample such as a surgical sample, e.g. of ocular cells/tissue.

In some embodiments, the polypeptide according to the present inventionmay be detected and/or distinguished from endogenous CR1 by Westernblotting, mass spectrometry and/or enzyme digestion, e.g. by a specificpeptidase. In some embodiments, the polypeptide may comprise a pointmutation to generate peptides by enzyme digestion that are distinct frompost-digestion peptides from endogenous CR1.

In some embodiments, a polypeptide according to the present inventionmay additionally comprise a cleavage site for removing a protein tag.For example, it may be desired to remove a tag used for purification ofthe polypeptide following purification. In some embodiments the cleavagesite may e.g. be a Tobacco Etch Virus (TEV) protease cleavage site, forexample as shown in SEQ ID NO:34.

In some embodiments a polypeptide according to the present inventioncomprises, or consists of, an amino acid sequence corresponding to SEQID NO:40, 42, 44, and/or 46. In some embodiments the polypeptidecomprises, or consists of, an amino acid sequence having at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:40, 42,44, and/or 46. Such polypeptides may have any length provided herein.

As used herein, a “polypeptide” includes molecules comprising more thanone polypeptide chain, which may be associated (e.g. covalently ornon-covalently) into a complex. That is, a “polypeptide” within themeaning of the present invention encompasses molecules comprising one ormore polypeptide chains. The polypeptide of the invention may in variousdifferent embodiments and at different stages of expression/productionin vitro or in vivo comprise e.g. a signal peptide, protein tag,cleavage sites for removal thereof, etc. The polypeptide of theinvention may comprise any CR1 CCP sequence described herein, or anycombination of CR1 CCP domains 8 (SEQ ID NO:8), 9 (SEQ ID NO:9), 10 (SEQID NO:10), 15 (SEQ ID NO:8), 16 (SEQ ID NO:11), and/or 17 (SEQ IDNO:12), or any combination of sequences ‘A’, ‘B’ and/or ‘C’ describedherein, optionally in combination with one or more of any of the furtherfeatures of the polypeptide of the invention described herein (e.g.signal peptide, linker, detection sequence, lack of glycosylation site,substituted amino acid residue, protein tag, cleavage site for removinga protein tag, secretory pathway sequence, cleavage site for removing asecretory pathway sequence).

Sequence Identity

As used herein, an amino acid sequence which corresponds to a referenceamino acid sequence may comprise at least 60%, e.g. one of at least 65%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to thereference sequence.

Pairwise and multiple sequence alignment for the purposes of determiningpercent identity between two or more amino acid or nucleic acidsequences can be achieved in various ways known to a person of skill inthe art, for instance, using publicly available computer software suchas ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee(Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign(Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT(Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780software. When using such software, the default parameters, e.g. for gappenalty and extension penalty, are preferably used.

Sequences

SEQ ID NO: Description Sequence 1 Human Complement Receptor 1 (UniProt:P17927; Entry version 181 (25 Oct. 2017), Sequence version 3 (02 Mar.2010)); residues 1-2039 Including signal sequence [CCP domains ‘8-10’and ‘15-17’ indicated by single underline, individual CCP domains 8, 9,10, 15, 16, 17 indicated by double underline; amino acid differencesbetween CCPs 8-10 and 15-17 indicated with wavy underline]MGASSPRSPEPVGPPAPGLPFCCGGSLLAVVVLLALPVAWGQCNAPEWLPFARPTNLTDEFEFPIGTYLNYECRPGYSGRPFSIICLKNSVWTGAKDRCRRKSCRNPPDPVNGMVHVIKGIQFGSQIKYSCTKGYRLIGSSSATCIISGDTVIWDNETPICDRIPCGLPPTITNGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEVVEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGYDLRGAASMRCTPQGDWSPAAPTCEVKSCDDFMGQLLNGRVLFPVNLQLGAKVDFVCDEGFQLKGSSASYCVLAGMESLWNSSVPVCEQIFCPSPPVIPNGRHTGKPLEVFPFGKTVNYTCDPHPDRGTSFDLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICORIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEVVEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGYDLRGAASMRCTPQGDWSPAAPTCEVKSCDDFMGQLLNGRVLFPVNLQLGAKVDFVCDEGFQLKGSSASYCVLAGMESLWNSSVPVCEQIFCPSPPVIPNGRHTGKPLEVFPFGKAVNYTCDPHPDRGTSFDLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRTPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSLYCTSNDDQVGTWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEVVEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPEILHGEHTPSHQDNFSPGQEVFYSCEPGYDLRGAASLHCTPQGDWSPEAPRCAVKSCDDFLGQLPHGRVLFPLNLQLGAKVSFVCDEGFRLKGSSVSHCVLVGMRSLWNNSVPVCEHIFCPNPPAILNGRHTGTPSGDIPYGKEISYTCDPHPDRGMTFNLIGESTIRCTSDPHGNGVWSSPAPRCELSVRAGHCKTPEQFPFASPTIPINDFEFPVGTSLNYECRPGYFGKMFSISCLENLVWSSVEDNCRRKSCGPPPEPFNGMVHINTDTQFGSTVNYSCNEGFRLIGSPSTTCLVSGNNVTWDKKAPICEIISCEPPPTISNGDFYSNNRTSFHNGTVVTYQCHTGPDGEQLFELVGERSIYCTSKDDQVGVWSSPPPRCISTNKCTAPEVENAIRVPGNRSFFSLTEIIRFRCQPGFVMVGSHTVQCQTNGRWGPKLPHCSRVCQPPPEILHGEHTLSHQDNFSPGQEVFYSCEPSYDLRGAASLHCTPQGDWSPEAPRCTVKSCDDFLGQLPHGRVLLPLNLQLGAKVSFVCDEGFRLKGRSASHCVLAGMKALWNSSVPVCEQIFCPNPPAILNGRHTGTPFGDIPYGKEISYACDTHPDRGMTFNLIGESSIRCTSDPQGNGVWSSPAPRCELSVPAACPHPPKIQNGHYIGGHVSLYLPGMTISYICDPGYLLVGKGFIFCTDQGIWSQLDHYCKEVNCSFPLFMNGISKELEMKKVYHYGDYVTLKCEDGYTLEGSPWSQCQADDRWDPPLAKCTSRTHDALIVGTLSGTIFFILLIIFLSWIILKHRKGNNAHENPKEVAIHLH SQGGSSVHPRTLQTNEENSRVLP2 Human Complement Receptor 1 CCPs 8-10 (UniProt: P17927 residues 491 to684) Without leader sequenceGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQC II 3 Human ComplementReceptor 1 CCPs 15-17 GHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHR (UniProt: P17927residues 941 to 1134) Without leader sequenceLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQC II 4 Human ComplementReceptor 1; consensus sequence for CCPs 8-10, 15-17 (UniProt: P17927residues 491 to 684; residues 941 to 1134) Without leader sequenceGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNX₁AHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNX₂GSX₃GRKVFELVGEPSIYCTSNDDQVGIWSGPAP QCII 5 HumanComplement Receptor 1 CCPs 8-10 (UniProt: P17927 residues 491 to 684)Non-glycosylated; Without leader sequence [mutated glycosylation sitesunderlined; substitutions in bold correspond to positions 509 and 578 ofUniProt: P179271 GHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQC II 6 Human ComplementReceptor 1 CCPs 15-17 (UniProt: P17927 residues 941 to 1134)Non-glycosylated; Without leader sequence [mutated glycosylation sitesunderlined; substitutions in bold correspond to positions 959 and 1028of UniProt: P179271 GHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQC II 7 Nativesignal/leader sequence from Factor H MRLLAKIICLMLWAICVA 8 HumanComplement Receptor 1 CCP8 /CCP15 (UniProt: P17927 residues491-550/941-1000) ‘Sequence A’GHCOAPDHFLFAKLKTOTNASDFPIGTSLKYECRPEYYGRPFSITCLD NLVWSSPKDVCKR 9 HumanComplement Receptor 1 CCP9 (UniProt: P17927 residues 552-612)KSCKTPPDPVNGMVHVITDIOVGSRINYSCTTGHRLIGHSSAECILSG NAAHWSTKPPICQ 10 HumanComplement Receptor 1 CCP10 (UniProt: P17927 residues 613-684RIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII 11 Human Complement Receptor 1 CCP16 (UniProt:P17927 residues 1002-1062)KSCKTPPDPVNGMVHVITDIOVGSRINYSCTTGHRLIGHSSAECILSG NTAHWSTKPPICO 12 HumanComplement Receptor 1 CCP17 (UniProt: P17927 residues 1063-1134RIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDOVGIWSGPAPQCII 13 Human ComplementGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLD Receptor 1 CCPs 8-10and 15-17 (contiguous; without leader sequence) [amino acid differencesbetween CCPs 8-10 and 15-17 indicated with wavy underline]NLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAP QCII 14 HumanComplement Receptor 1 CCPs 8-10 (linker) 15-17GHCOAPDHFLFAKLKTOTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIOVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICORIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII-[linker]-GHCOAPDHFLFAKLKTOTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIOVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQC II 15 Human ComplementReceptor 1 CCPs 8-10 and 15-17 (contiguous) Non-glycosylated; Withoutleader sequence [Mutated glycosylation sites underlined; substitutionsin bold correspond to positions 509, 578, 959 and 1028 of UniProt:P179271 GHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAP QCII 16 HumanComplement Receptor 1; Consensus sequence for CCPs 9, 16 (correspondingto UniProt: P17927 residues 552-612 and 1002-1062) ‘Sequence B’KSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSG NX₁AHWSTKPPICQ 17 HumanComplement Receptor 1; Consensus sequence for CCPs 10, 17 (correspondingto (UniProt: P17927 residues 613-684 and 1063-1134) ‘Sequence C’RIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNX₂GSX₃GRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII 18 Human C3 (UniProt: P01024; Entry version221 (20 Dec. 2017); Sequence version 2 (12 Dec. 2006)) including signalpeptide MGPTSGPSLLLLLLTHLPLALGSPMYSIITPNILRLESEETMVLEAHDAQGDVPVTVTVHDFPGKKLVLSSEKTVLTPATNHMGNVTFTIPANREFKSEKGRNKFVTVQATFGTQVVEKVVLVSLQSGYLFIQTDKTIYTPGSTVLYRIFTVNHKLLPVGRTVMVNIENPEGIPVKQDSLSSQNQLGVLPLSWDIPELVNMGQWKIRAYYENSPQQVFSTEFEVKEYVLPSFEVIVEPTEKFYYIYNEKGLEVTITARFLYGKKVEGTAFVIFGIQDGEQRISLPESLKRIPIEDGSGEVVLSRKVLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAERSGIPIVTSPYQIHFTKTPKYFKPGMPFDLMVFVTNPDGSPAYRVPVAVQGEDTVQSLTQGDGVAKLSINTHPSQKPLSITVRTKKQELSEAEQATRTMQALPYSTVGNSNNYLHLSVLRTELRPGETLNVNFLLRMDRAHEAKIRYYTYLIMNKGRLLKAGRQVREPGQDLVVLPLSITTDFIPSFRLVAYYTLIGASGQREVVADSVWVDVKDSCVGSLVVKSGQSEDRQPVPGQQMTLKIEGDHGARVVLVAVDKGVFVLNKKNKLTQSKIWDVVEKADIGCTPGSGKDYAGVFSDAGLTFTSSSGQQTAQRAELQCPQPAARRRRSVQLTEKRMDKVGKYPKELRKCCEDGMRENPMRFSCQRRTRFISLGEACKKVFLDCCNYITELRRQHARASHLGLARSNLDEDIIAEENIVSRSEFPESWLWNVEDLKEPPKNGISTKLMNIFLKDSITTWEILAVSMSDKKGICVADPFEVTVMQDFFIDLRLPYSVVRNEQVEIRAVLYNYRQNQELKVRVELLHNPAFCSLATTKRRHQQTVTIPPKSSLSVPYVIVPLKTGLQEVEVKAAVYHHFISDGVRKSLKVVPEGIRMNKTVAVRTLDPERLGREGVQKEDIPPADLSDQVPDTESETRILLQGTPVAQMTEDAVDAERLKHLIVTPSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSRSSKITHRIHWESASLLRSEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLAFKVHQYFNVELIQPGAVKVYAYYNLEESCTRFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEHWPEEDECQDEENQKQCQDLGAFTESMVVFGCPN 19 Human C3 β chain (UniProt: P01024;Entry version 221 (20 Dec. 2017); 20 Sequence version 2 (12 Dec. 2006);residues 23-667) SPMYSIITPNILRLESEETMVLEAHDAQGDVPVTVTVHDFPGKKLVLSSEKTVLTPATNHMGNVTFTIPANREFKSEKGRNKFVTVQATFGTQVVEKVVLVSLQSGYLFIQTDKTIYTPGSTVLYRIFTVNHKLLPVGRTVMVNIENPEGIPVKQDSLSSQNQLGVLPLSWDIPELVNMGQWKIRAYYENSPQQVFSTEFEVKEYVLPSFEVIVEPTEKFYYIYNEKGLEVTITARFLYGKKVEGTAFVIFGIQDGEQRISLPESLKRIPIEDGSGEVVLSRKVLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAERSGIPIVTSPYQIHFTKTPKYFKPGMPFDLMVFVTNPDGSPAYRVPVAVQGEDTVQSLTQGDGVAKLSINTHPSQKPLSITVRTKKQELSEAEQATRTMQALPYSTVGNSNNYLHLSVLRTELRPGETLNVNFLLRMDRAHEAKIRYYTYLIMNKGRLLKAGRQVREPGQDLVVLPLSITTDFIPSFRLVAYYTLIGASGQREVVADSVWVDVKDSCVGSLVVKSGQSEDRQPVPGQQMTLKIEGDHGARVVLVAVDKGVFVLNKKNKLTQSKIWDVVEKADIGCTPGSGKDYAGVFSDAGLTF TSSSGQQTAQRAELQCPQPAA20 Human C3 α′ chain (UniProt: P01024; Entry version 221 (20 Dec. 2017);Sequence version 2 (12 Dec. 2006); residues 749-1663)SNLDEDIIAEENIVSRSEFPESWLWNVEDLKEPPKNGISTKLMNIFLKDSITTWEILAVSMSDKKGICVADPFEVTVMQDFFIDLRLPYSVVRNEQVEIRAVLYNYRQNQELKVRVELLHNPAFCSLATTKRRHQQTVTIPPKSSLSVPYVIVPLKTGLQEVEVKAAVYHHFISDGVRKSLKVVPEGIRMNKTVAVRTLDPERLGREGVQKEDIPPADLSDQVPDTESETRILLQGTPVAQMTEDAVDAERLKHLIVTPSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSRSSKITHRIHWESASLLRSEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLAFKVHQYFNVELIQPGAVKVYAYYNLEESCTRFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEHWPEEDECQDEENQKQCQDLGAFTESMVVFG CPN 21 Human C3a(UniProt: P01024; Entry version 221 (20 Dec. 2017); Sequence version 2(12 SVQLTEKRMDKVGKYPKELRKCCEDGMRENPMRFSCQRRTRFISLGEACKKVFLDCCNYITELRRQHARASHLGLAR December 2006); residues 672-748) 22 HumanC3 α′ chain fragment 1 (UniProt: P01024; Entry version 221 (20 Dec.2017); Sequence version 2 (12 Dec. 2006); residues 749-1303)SNLDEDIIAEENIVSRSEFPESWLWNVEDLKEPPKNGISTKLMNIFLKDSITTWEILAVSMSDKKGICVADPFEVTVMQDFFIDLRLPYSVVRNEQVEIRAVLYNYRQNQELKVRVELLHNPAFCSLATTKRRHQQTVTIPPKSSLSVPYVIVPLKTGLQEVEVKAAVYHHFISDGVRKSLKVVPEGIRMNKTVAVRTLDPERLGREGVQKEDIPPADLSDQVPDTESETRILLQGTPVAQMTEDAVDAERLKHLIVTPSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAPDHQELNLDVSLQLPSR 23 Human C3 α′ chain fragment 2 (UniProt:P01024; ; Entry version 221 (20 Dec. 2017); Sequence version 2 (12 Dec.2006); residues 1321-1663) Also known as C3c fragment 2SEETKENEGFTVTAEGKGQGTLSVVTMYHAKAKDQLTCNKFDLKVTIKPAPETEKRPQDAKNTMILEICTRYRGDQDATMSILDISMMTGFAPDTDDLKQLANGVDRYISKYELDKAFSDRNTLIIYLDKVSHSEDDCLAFKVHQYFNVELIQPGAVKVYAYYNLEESCTRFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSGSDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEHWPEEDECQDEENQKQCQDLGAFTESM VVFGCPN 24 Human C3f(UniProt: P01024; ; Entry version 221 (20 Dec. 2017); Sequence version 2(12 Dec. 2006); residues 1304-1320) SSKITHRIHWESASLLR 25 HumanComplement Factor | (UniProt: P05156; Entry version 192 (20 Dec. 2017)Sequence version 2 (11 Jan. 2011))MKLLHVFLLFLCFHLRFCKVTYTSQEDLVEKKCLAKKYTHLSCDKVFCQPWQRCIEGTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLECLHPGTKFLNNGTCTAEGKFSVSLKHGNTDSEGIVEVKLVDQDKTMFICKSSWSMREANVACLDLGFQQGADTQRRFKLSDLSINSTECLHVHCRGLETSLAECTFTKRRTMGYQDFADVVCYTQKADSPMDDFFQCVNGKYISQMKACDGINDCGDQSDELCCKACQGKGFHCKSGVCIPSQYQCNGEVDCITGEDEVGCAGFASVTQEETEILTADMDAERRRIKSLLPKLSCGVKNRMHIRRKRIVGGKRAQLGDLPWQVAIKDASGITCGGIYIGGCWILTAAHCLRASKTHRYQIWTTVVDWIHPDLKRIVIEYVDRIIFHENYNAGTYQNDIALIEMKKDGNKKDCELPRSIPACVPWSPYLFQPNDTCIVSGWGREKDNERVFSLQWGEVKLISNCSKFYGNRFYEKEMECAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGVVSWGENCGKPEFPGVYTKVANYFDWISYHVGRP FISQYNV 26 HumanComplement Factor I proteolytic domain (UniProt: P05156; Entry version192 (20 Dec. 2017) Sequence version 2 (11 Jan. 2011); residues 340-574)IVGGKRAQLGDLPWQVAIKDASGITCGGIYIGGCWILTAAHCLRASKTHRYQIWTTVVDWIHPDLKRIVIEYVDRIIFHENYNAGTYQNDIALIEMKKDGNKKDCELPRSIPACVPWSPYLFQPNDTCIVSGWGREKDNERVFSLQWGEVKLISNCSKFYGNRFYEKEMECAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGVVSWGENCGKPEFPGVYTKVANYFDWISYHVG 27 Consensus sequence forN-linked glycosylation NX₁X₂ wherein X₁ = any amino acid except for P X₂= S or T 28 Complement Factor H isoform FHL-1 (UniProt: P08603-2)Including leader sequence (underlined) SEQ ID No. 7MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLY 29 Human ComplementFactor H (UniProt: P08603; Entry version 214 (25 Oct. 2017) Sequenceversion 4 (11 Sep. 2007)) Including leader sequence (underlined) SEQ IDNo. 7 MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRVKTCSKSSIDIENGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIKSCDIPVFMNARTKNDFTWFKLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPICYERECELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPICKEQVQSCGPPPELLNGNVKEKTKEEYGHSEVVEYYCNPRFLMKGPNKIQCVDGEWTTLPVCIVEESTCGDIPELEHGWAQLSSPPYYYGDSVEFNCSESFTMIGHRSITCIHGVWTQLPQCVAIDKLKKCKSSNLIILEEHLKNKKEFDHNSNIRYRCRGKEGWIHTVCINGRWDPEVNCSMAQIQLCPPPPQIPNSHNMTTTLNYRDGEKVSVLCQENYLIQEGEEITCKDGRWQSIPLCVEKIPCSQPPQIEHGTINSSRSSQESYAHGTKLSYTCEGGFRISEENETTCYMGKWSSPPQCEGLPCKSPPEISHGVVAHMSDSYQYGEEVTYKCFEGFGIDGPAIAKCLGEKWSHPPSCIKTDCLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNVTCINSRWTGRPTCRDTSCVNPPTVQNAYIVSRQMSKYPSGERVRYQCRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYSRTGESVEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR 30 Human Complement Receptor 1 (UniProt:P17927 residues 491 to 1134, from CCP8 to CCP17) [CCP domains ‘8-10’ and‘15-17’ indicated by single underline, individual CCP domains 8, 9, 10,15, 16, 17 indicated by double underline; amino acid differences betweenCCPs 8-10 and 15-17 indicated in bold/wavy underline]GHCOAPDHFLFAKLKTOTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIOVGSRINYSCTTGHPLIGHSSAECILSGNAAHWSTKPPICORIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEVVEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGYDLRGAASMRCTPQGDWSPAAPTCEVKSCDDFMGQLLNGRVLFPVNLQLGAKVDFVCDEGFQLKGSSASYCVLAGMESLWNSSVPVCEQIFCPSPPVIPNGRHTGKPLEVFPFGKAVNYTCDPHPDRGTSFDLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIY CTSNDDQVGIWSGPAPQCII 31Human Complement Receptor 1 (UniProt: P17927 residues 491 to 1134, CCP8to CCP17) Non-glycosylated [CCP domains ‘8-10’ and ‘15-17’ indicated bysingle underline, individual CCP domains 8, 9, 10, 15, 16, 17 indicatedby double underline; amino acid differences between CCPs 8-10 and 15-17indicated in bold/wavy underline; substitutions inGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICORIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEVVEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGYDLRGAASMRCTPQGDWSPAAPTCEVKSCDDFMGQLLNGRVLFPVNLQLGAKVDFVCDEGFQLKGSSASYCVLAGMESLWNSSVPVCEQIFCPSPPVIPNGRHTGKPLEVFPFGKAVNYTCDPHPDRGTSFDLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIY CTSNDDOVGTWSGPAPQCIIbold/dotted underline correspond to positions 509, 578, 959 and 1028 ofUniProt: P17927]] 32 Human Complement Receptor 1 (UniProt: P17927; Entryversion 181 (25 Oct. 2017), Sequence version 3 (02 Mar. 2010))Transmembrane domain (residues 1972-1996 ALIVGTLSCGTIFFILLIIFLSWIIL 33Human Complement Receptor 1 (UniProt: P17927; Entry version 181 (25 Oct.2017), Sequence version 3 (02 Mar. 2010)) Cytoplasmic tail (residues1997-2039) KHRKGNNAHENPKEVAIHLHSQGGSSVHPRTLQTNEENSRVLP 34 Tobacco EtchVirus (TEV) cleavage site ENLYFQGS 35 Human Complement Receptor 1 CCPs8-10 protein coding sequence Including leader sequence (doubleunderlined) Including HindIll restriction enzyme cleavage site (AAGCTT),Kozak consensus sequence (GCCACC), stop codon (TAA) and EcoRVrestriction enzyme cleavage site (GATATC)AAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCGGACATTGTCAGGCCCCTGACCACTTCCTGTTCGCCAAGCTGAAAACCCAGACCAACGCCAGCGACTTCCCTATCGGCACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCAACTACAGCTGCACCACCGGCCACAGACTGATCGGACACTCTAGCGCCGAGTGTATCCTGAGCGGCAATGCCGCACACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAATCCTGGCAGCGGCGGCAGAAAGGTGTTCGAACTTGTGGGCGAGCCCAGCATCTACTGCACCAGCAACGATGACCAAGTCGGCATTTGGAGCGGCCCTGCTCCTCAGTGCATCATCTAAGATATC 36 Human Complement Receptor 1 CCPs8-10 protein coding sequence (underlined) Non-glycosylated Includingleader sequence (double underlined) Including HindIll restriction enzymecleavage site (AAGCTT), Kozak consensus sequence (GCCACC), stop codon(TAA) and EcoRV restriction enzyme cleavage site (GATATC)AAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCGGACATTGTCAGGCCCCTGACCACTTCCTGTTCGCCAAGCTGAAAACCCAGACACAGGCCAGCGACTTCCCTATCGGCACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCCAGTACAGCTGCACCACAGGCCACAGACTGATCGGCCACTCTAGCGCCGAGTGTATCCTGTCTGGCAATGCCGCTCACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAATCCTGGCAGCGGCGGCAGAAAGGTGTTCGAACTTGTGGGCGAGCCCAGCATCTACTGCACCAGCAACGATGACCAAGTCGGCATTTGGAGCGGCCCTGCTCCTCAGTGCATCATCTAAGATATC 37 Human Complement Receptor 1 CCPs15-17 protein coding sequenceAAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCGGCCACTGTCAGGCCCCTGATCACTTCCTGTTCGCCAAGCTGAAAACCCAGACCAACGCCAGCGACTTCCCTATCGGC (underlined) Includingleader sequence (double underlined) Including HindIll restriction enzymecleavage site (AAGCTT), Kozak consensus sequence (GCCACC), stop codon(TAA) and EcoRV restriction enzyme cleavage site (GATATC)ACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCAACTACAGCTGCACCACCGGCCACAGACTGATCGGACACTCTAGCGCCGAGTGTATCCTGAGCGGCAATGCCGCACACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAATCCTGGCAGCGGCGGCAGAAAGGTGTTCGAACTTGTGGGCGAGCCCAGCATCTACTGCACCAGCAACGATGACCAAGTCGGCATTTGGAGCGGCCCTGCTCCTCAGTGCATCATCCCTAAGATATC 38 Human Complement Receptor 1 CCPs15-17 protein coding sequence (underlined) Non-glycosylated Includingleader sequence (double underlined) Including HindIll restriction enzymecleavage site (AAGCTT), Kozak consensus sequence (GCCACC), stop codon(TAA) and EcoRV restriction enzyme cleavage site (GATATC)AAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCGGCCACTGTCAGGCCCCTGATCACTTCCTGTTCGCCAAGCTGAAAACCCAGACACAGGCCAGCGACTTCCCTATCGGCACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCCAGTACAGCTGCACCACAGGCCACAGACTGATCGGCCACTCTAGCGCCGAGTGTATCCTGAGCGGAAACACAGCCCACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAACCTGGGCTCCAGAGGCCGGAAGGTGTTCGAACTTGTGGGCGAGCCTAGCATCTACTGCACCAGCAACGACGACCAAGTCGGCATTTGGAGCGGACCTGCTCCTCAGTGCATCATCCCTAAGATATC 39 Human Complement Receptor 1 CCPs8-10 protein coding sequence (underlined) Including leader sequence(doubled underlined); His tag (dotted line); TEV cleavage site (dashedline) Including HindIll restriction enzyme cleavage site (AAGCTT), Kozakconsensus sequence (GCCACC), stop codon (TAA) and EcoRV restrictionenzyme cleavage site (GATATC)AAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCCACCACCATCACCATCACGGCAGCAGCGAGAACCTGTACTTCCAAGGATCTTCTGGCGGCCACTGTCAGGCCCCTGATCACTTCCTGTTCGCCAAGCTGAAAACCCAGACCAACGCCAGCGACTTCCCTATCGGCACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCAACTACAGCTGCACCACCGGCCACAGACTGATCGGACACTCTAGCGCCGAGTGTATCCTGAGCGGCAATGCCGCACACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAATCCTGGCAGCGGCGGCAGAAAGGTGTTCGAACTTGTGGGCGAGCCCAGCATCTACTGCACCAGCAACGATGACCAAGTCGGCATTTGGAGCGGCCCTGCTCCTCAGTGCATCATCTAAGATATCMRLLAKIICLMLWAICVAHHHHHHGSSENLYFQGSSGGHCQAPDHFLF 40 Human ComplementReceptor 1 CCPs 8-10 Including leader sequence (doubled underlined); Histag (dotted line); TEV cleavage site (dashed line)MRLLAKIICLMLWAICVAHHHHHHGSSENLYFQGSSGGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII 41 Human ComplementAAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTG Receptor 1 CCPs 8-10protein coding sequence (underlined) Non-glycosylated Including leadersequence (doubled underlined); His tag (dotted line); TEV cleavage site(dashed line) Including HindIll restriction enzyme cleavage site(AAGCTT), Kozak consensus sequence (GCCACC), stop codon (TAA) and EcoRVrestriction enzyme cleavage site (GATATC)TGGGCCATCTGCGTGGCCCACCACCATCACCATCACGGCAGCAGCGAGAACCTGTACTTCCAAGGATCTTCTGGCGGCCACTGTCAGGCCCCTGATCACTTCCTGTTCGCCAAGCTGAAAACCCAGACACAGGCCAGCGACTTCCCTATCGGCACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCCAGTACAGCTGCACCACAGGCCACAGACTGATCGGCCACTCTAGCGCCGAGTGTATCCTGTCTGGCAATGCCGCTCACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAATCCTGGCAGCGGCGGCAGAAAGGTGTTCGAACTTGTGGGCGAGCCCAGCATCTACTGCACCAGCAACGATGACCAAGTCGGCATTTGGAGCGGCCCTGCTCCTCAGTGCATCATCTAAGATATC 42 Human Complement Receptor1 CCPs 8-10 Non-glycosylated Including leader sequence (doubledunderlined); His tag (dotted line); TEV cleavage site (dashed line)MRLLAKIICLMLWAICVARHHHHHHGSSENLYFQGSSGGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII 43 Human Complement Receptor 1CCPs 15-17 protein coding sequence (underlined) Including leadersequence (doubled underlined); His tag (dotted line); TEV cleavage site(dashed line) Including HindIll restriction enzyme cleavage site(AAGCTT), Kozak consensus sequence (GCCACC), stop codon (TAA) and EcoRVrestriction enzyme cleavage site (GATATC)AAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCCACCACCATCACCATCACGGCAGCAGCGAGAACCTGTACTTCCAAGGATCTTCTGGCGGCCACTGTCAGGCCCCTGATCACTTCCTGTTCGCCAAGCTGAAAACCCAGACCAACGCCAGCGACTTCCCTATCGGCACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCAACTACAGCTGCACCACCGGCCACAGACTGATCGGACACTCTAGCGCCGAGTGTATCCTGAGCGGCAATGCCGCACACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAATCCTGGCAGCGGCGGCAGAAAGGTGTTCGAACTTGTGGGCGAGCCCAGCATCTACTGCACCAGCAACGATGACCAAGTCGGCATTTGGAGCGGCCCTGCTCCTCAGTGCATCATCCCTAAGATATCMRLLAKIICLMLWAICVAHHHHHHGSSENLFQGSSGGHCQAPDHFLF 44 Human ComplementReceptor 1 CCPs 15-17 Including leader sequence (doubled underlined);His tag (dotted line); TEV cleavage site (dashed line)MRLLAKIICLMLWAICVAHHHHHHGSSENLYFQGSSGGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII 45 Human Complement Receptor 1CCPs 15-17 AAGCTTGCCACCATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCCACCACCATCACCATCACGGCAGCAGCGAG protein coding sequence(underlined) Non-glycosylated Including leader sequence (doubledunderlined); His tag (dotted line); TEV cleavage site (dashed line)Including HindIll restriction enzyme cleavage site (AAGCTT), Kozakconsensus sequence (GCCACC), stop codon (TAA) and EcoRV restrictionenzyme cleavage site (GATATC)AACCTGTACTTCCAAGGATCTTCTGGCGGCCACTGTCAGGCCCCTGATCACTTCCTGTTCGCCAAGCTGAAAACCCAGACACAGGCCAGCGACTTCCCTATCGGCACCAGCCTGAAGTACGAGTGCAGACCCGAGTACTACGGCAGACCCTTCAGCATCACCTGTCTGGACAACCTCGTGTGGTCTAGCCCCAAGGACGTGTGCAAGAGAAAGAGCTGCAAGACCCCTCCTGATCCTGTGAACGGCATGGTGCACGTGATCACCGACATCCAAGTGGGCAGCAGAATCCAGTACAGCTGCACCACAGGCCACAGACTGATCGGCCACTCTAGCGCCGAGTGTATCCTGAGCGGAAACACAGCCCACTGGTCCACCAAGCCTCCAATCTGCCAGAGAATCCCTTGCGGCCTGCCTCCTACAATCGCCAACGGCGATTTCATCAGCACCAACAGAGAGAACTTCCACTACGGCTCCGTGGTCACCTACAGATGCAACCTGGGCTCCAGAGGCCGGAAGGTGTTCGAACTTGTGGGCGAGCCTAGCATCTACTGCACCAGCAACGACGACCAAGTCGGCATTTGGAGCGGACCTGCTCCTCAGTGCATCATCCCTAAGATATC 46 Human ComplementReceptor 1 CCPs 15-17 Non-glycosylated Including leader sequence(doubled underlined); His tag (dotted line); TEV cleavage site (dashedline) MRLLAKIICLMLWAICVAHHHHHHHGSSENLYFQGSSGGHCQAPDHFLFMRLLAKIICLMLWAICVAHHHHHHGSSENLYFQGSSGGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII 47 CR1a Including leadersequence (double underline); CCPs 8-10MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIY CTSNDDQVGIWSGPAPQCII 48nCR1a Including leader sequence (double underline); CCPs 8-10; N509Q andN578Q (numbered according to UniProt: P17927)MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIY CTSNDDQVGIWSGPAPQCII 49CR1b Including leader sequence (double underline); CCPs 15-17MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIY CTSNDDQVGIWSGPAPQCII 50nCR1b Including leader sequence (double underline); CCPs 15-17; N959Qand N1028Q (numbered according to UniProt: P17927)MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIY CTSNDDQVGIWSGPAPQCII 51CR1a+CR1b Including leader sequence (double underline); CCPs 8-10 +MRLLAKIICLMLWAICVAGHCQAPDHEFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIY 52 nCR1a+nCR1bIncluding leader sequence (double underline); CCPs 8-10 + 15-17; N509Qand N578Q, N959Q and N1028Q (numbered according to UniProt: P17927)MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPS IYCTSNDDQVGIWSGPAPQCII53 CR1a+nCR1b Including leader sequence (double underline); CCPs 8-10 +15-17; N959Q and N1028Q (numbered according to UniProt: P17927)MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPS IYCTSNDDQVGIWSGPAPQCII54 nCR1a+CR1b Including leader sequence (double underline); CCPs 8-10 +15-17; N509Q and N578Q (numbered according to UniProt: P17927)MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNTAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPS IYCTSNDDQVGIWSGPAPQCII

Functional Properties of the Polypeptide

The polypeptide according to the present invention may be characterisedby reference to one or more functional properties.

In particular, a polypeptide according to the present invention maypossess one or more of the following properties (as determined byanalysis in an appropriate assay for said property):

-   Binds to C3b;-   Binds to C3b with an affinity of binding which is similar to the    affinity of binding to C3b displayed by a co-factor for Complement    Factor I (or a fragment thereof);-   Binds to C3b with an affinity of binding which is higher than the    affinity of binding to C3b displayed by a co-factor for Complement    Factor I (or a fragment thereof);-   Binds to C3b with an affinity of binding which is similar to the    affinity of binding to C3b displayed by Complement Receptor 1 (or a    fragment thereof);-   Binds to C3b in the region of C3b bound by a co-factor for    Complement Factor I (or a fragment thereof);-   Binds to C3b in the region of C3b bound by Complement Receptor 1 (or    a fragment thereof);-   Binds to C3b in the region of C3b bound by Complement Receptor 1 CCP    domains 8-10 and/or 15-17 (or a fragment thereof);-   Acts as a co-factor to enable Complement Factor I-mediated    inactivation of C3b;-   Acts as a co-factor to enable Complement Factor I-mediated    reduction/prevention of the formation of a functional C3bBb-type C3    convertase;-   Acts as a co-factor to enable Complement Factor I-mediated    reduction/prevention of the formation of a functional C3bBb3b-type    C5 convertase;-   Acts as a co-factor to enable Complement Factor I-mediated    reduction/prevention of the formation of a functional C4b2a3b-type    C5 convertase;-   Acts as a co-factor to enable Complement Factor I-mediated reduction    of C3bBb-type C3 convertase activity;-   Acts as a co-factor to enable Complement Factor I-mediated reduction    of C3bBb3b-type C5 convertase activity;-   Acts as a co-factor to enable Complement Factor I-mediated reduction    of C4b2a3b-type C5 convertase activity;-   Acts as a co-factor to enable Complement Factor I-mediated reduction    of the amount of C3bBb-type C3 convertase;-   Acts as a co-factor to enable Complement Factor I-mediated reduction    of the amount of C3bBb3b-type C5 convertase;-   Acts as a co-factor to enable Complement Factor I-mediated reduction    of the amount of C4b2a3b-type C5 convertase;-   Reduces the amount of C3b via Complement Factor I;-   Increases the amount of iC3b via Complement Factor I;-   Increases the amount of C3dg via Complement Factor I;-   Increases the amount of C3d via Complement Factor I;-   Increases the amount of C3f via Complement Factor I;-   Reduces the amount of C5b via Complement Factor I;-   Reduces the amount of C5a via Complement Factor I;-   Decreases the amount of iC3b via Complement Factor I compared to the    amount of iC3b produced by FH and/or FHL-1 via Complement Factor I;-   Increases the ratio of C3dg to iC3b via Complement Factor I;-   Is capable of inhibiting complement activation;-   Diffuses through Bruch’s membrane (BrM);-   Displays superior ability to diffuse through BrM compared to    Complement Factor I;-   Displays superior ability to diffuse through BrM compared to a    co-factor for Complement Factor I (or a fragment thereof);-   Displays similar ability to diffuse through BrM compared to a    co-factor for Complement Factor I (or a fragment thereof);-   Displays superior ability to diffuse through BrM compared to    Complement Factor H;-   Displays similar ability to diffuse through BrM compared to    Complement Factor H isoform FHL-1;-   Displays superior ability to diffuse through BrM compared to    Complement Factor H isoform FHL-1;-   Displays similar ability to diffuse through BrM compared to soluble    Complement Receptor 1;-   Displays superior ability to diffuse through BrM compared to soluble    Complement Receptor 1.

Whether a given polypeptide possesses the functional properties referredto in the previous paragraph can be analysed, for example, as describedherein.

A polypeptide according to the present invention may be capable ofbinding to C3b. In some embodiments, a polypeptide according to thepresent invention may comprise or consist of a C3b binding region. Insome embodiments, the C3b binding region of the polypeptide according tothe present invention comprises or consists of a C3b binding region ofCR1, e.g. CCP domains 8-10 and/or 15-17.

As used herein, a “C3b binding region” refers to a region capable ofbinding to C3b. In some embodiments, the C3b binding region is capableof specific binding to C3b. Binding to C3b may be mediated bynon-covalent interactions such as Van der Waals forces, electrostaticinteractions, hydrogen bonding, and hydrophobic interactions formedbetween the C3b binding region and C3b. In some embodiments, the C3bbinding region binds to C3b with greater affinity, and/or with greaterduration than it binds to molecules other than C3b.

The ability of a polypeptide according to the present invention or aputative C3b binding region to bind to C3b can be analysed usingtechniques well known to the person skilled in the art, including ELISA,Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol(2012) 907:411-442; or Rich et al., Anal Biochem. 2008 Feb 1;373(1):112-20), Bio-Layer Interferometry (see e.g. Lad et al., (2015) JBiomol Screen 20(4): 498-507; or Concepcion et al., Comb Chem HighThroughput Screen. 2009 Sep; 12(8):791-800), MicroScale Thermophoresis(MST) analysis (see e.g. Jerabek-Willemsen et al., Assay Drug DevTechnol. 2011 Aug; 9(4): 342-353), or by a radiolabelled antigen bindingassay (RIA). Through such analysis binding to a given target can bedetermined and quantified. In some embodiments, the binding may be theresponse detected in a given assay.

In some embodiments, a polypeptide according to the present inventiondisplays binding to C3b in such an assay which is greater than 1 times,e.g. oneof >1.01, >1.02, >1.03, >1.04, >1.05, >1.06, >1.07, >1.08, >1.09, >1.1, >1.2, >1.3, >1.4, >1.5, >1.6, >1.7, >1.8, >1.9, >2, >3, >4, >5, >6, >7, >8, >9, >10, >15, >20, >25, >30, >35, >40, >45, >50, >60, >70, >80, >90,or >100 times the level of binding signal detected in such an assay to anegative control molecule to which the C3b binding region does not bind.

In some embodiments, a polypeptide according to the present invention iscapable of binding to C3b with an affinity of binding which is similarto the affinity of binding to C3b displayed by CR1 or another co-factorfor Complement Factor I (or a fragment thereof) in a given assay. Anaffinity of binding which is similar to a reference affinity of bindingcan be e.g. ±40% of the level of binding, e.g. one of ±35%, ±30%, ±25%,±20%,±15%, ±10% or ±5% of the level of binding to C3b displayed byreference CR1 or the reference co-factor for Complement Factor I in acomparable assay.

In some embodiments a polypeptide according to the present invention iscapable of binding to C3b with an affinity of binding which is higherthan the affinity of binding to C3b displayed by a co-factor forComplement Factor I (or a fragment thereof) in a given assay. In someembodiments a polypeptide according to the present invention is capableof binding to C3b with an affinity of binding which is 1.5 times, 2times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9times, 9.5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35times, 40 times, 45 times, 50 times, 75 times, 100 times, 150 times, 200times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times,550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850times, 900 times, 950 times, or 1000 times the affinity of binding toC3b displayed by a co-factor for Complement Factor I (or a fragmentthereof) in a given assay. In some embodiments a polypeptide accordingto the present invention is capable of binding to C3b with an affinityof binding which is 10000 times, 100000 times, or 1000000 times theaffinity of binding to C3b displayed by a co-factor for ComplementFactor I (or a fragment thereof) in a given assay. In some embodiments apolypeptide according to the present invention is capable of binding toC3b with an affinity of binding which is 2, 3, 4, 5, 6, 7, 8, 9 or 10order(s) of magnitude greater than the affinity of binding to C3bdisplayed by a co-factor for Complement Factor I (or a fragment thereof)in a given assay. In some embodiments the co-factor for ComplementFactor I is Complement Factor H or truncated FH isoform FHL-1. Theco-factor for Complement Factor I may be CR1.

In some embodiments, a polypeptide according to the present invention iscapable of binding to C3b in the region of C3b that is bound by aco-factor for Complement Factor I (i.e. binds to the same region or anoverlapping region). In some embodiments, the polypeptide is capable ofbinding to C3b in the region bound by CR1 (or a fragment thereof). Insome embodiments, the polypeptide is capable of binding to C3b in theregion bound by CR1 CCP domains 8-10 and/or 15-17. In some embodiments,the polypeptide is capable of binding to C3b in the region bound by oneor more of Complement Factor I co-factors Complement Factor H, CD46,CD55, C4BP, SPICE, VCP, or MOPICE (or fragments thereof).

Whether a polypeptide according to the present invention binds to C3b inthe region of C3b bound by a given co-factor for Complement Factor I (ora fragment thereof) can be determined by various methods known to theskilled person, including ELISA, and surface plasmon resonance (SPR)analysis. An example of a suitable assay to determine whether a C3bbinding region binds to C3b in the region bound by a given co-factor forComplement Factor I (or a fragment thereof) is a competition ELISAassay.

For example, whether a polypeptide according to the present inventionbinds to C3b in the region of C3b bound by a given co-factor forComplement Factor I (or a fragment thereof) can be determined byanalysis of interaction of the co-factor/fragment with C3b in thepresence of, or following incubation of one or both of theco-factor/fragment and C3b with the polypeptide according to the presentinvention. A C3b binding region which binds to C3b in the region of C3bbound by a given co-factor/fragment is identified by the observation ofa reduction/decrease in the level of interaction between theco-factor/fragment and C3b in the presence of — or following incubationof one or both of the interaction partners with - the polypeptideaccording to the present invention, as compared to the level ofinteraction in the absence of the polypeptide according to the presentinvention (or in the presence of an appropriate controlpeptide/polypeptide). Suitable analysis can be performed in vitro, e.g.using recombinant interaction partners. For the purposes of such assays,one or both of the interaction partners and/or the polypeptide accordingto the present invention may be labelled, or used in conjunction with adetectable entity for the purposes of detecting and/or measuring thelevel of interaction.

In some embodiments, the polypeptide according to the present inventionacts as a co-factor for Complement Factor l. For example, thepolypeptide may potentiate cleavage of C3b by Complement Factor I,and/or present C3b in a favourable orientation for proteolytic cleavageby Complement Factor l. The polypeptide preferably does not inhibitproteolytic cleavage of C3b by Complement Factor l. In some embodiments,the Complement Factor I is endogenous. In some embodiments, theComplement Factor I is exogenous.

As used herein, an ‘endogenous’ protein/peptide refers to aprotein/peptide which is encoded/expressed by the relevant cell type,tissue, or subject (prior to treatment with a polypeptide, nucleic acid,vector, cell or pharmaceutical composition according to the presentinvention). A ‘non-endogenous’ or ‘exogenous’ protein/peptide refers toa protein/peptide which is not encoded/expressed by, the relevant celltype, tissue, or subject (prior to treatment with a polypeptide, nucleicacid, vector, cell or pharmaceutical composition according to thepresent invention).

A polypeptide according to the present invention that acts as aco-factor for Complement Factor I can be determined by e.g. by analysisof the level or rate of proteolytic cleavage of C3b by Complement FactorI in a suitable assay in the presence of (or after incubation with) apolypeptide according to the present invention as compared to the levelor rate of proteolytic cleavage of C3b by Complement Factor I in theabsence of the polypeptide according to the present invention (or in thepresence of an appropriate control peptide/polypeptide). A C3b bindingregion which acts as a co-factor for Complement Factor I is identifiedby the detection of an increased level or rate of proteolytic cleavageof C3b by Complement Factor I in the presence of (or after incubationwith) a polypeptide according to the present invention as compared tothe level or rate of proteolytic cleavage of C3b by Complement Factor Iin the absence of the polypeptide according to the present invention (orin the presence of an appropriate control peptide/polypeptide). Thelevel or rate of proteolytic cleavage of C3b by Complement Factor I canbe determined e.g. by detection of one or more products of cleavage ofC3b by Complement Factor I, e.g. iC3b, C3dg, C3d or C3f. The level orrate of proteolytic cleavage of C3b by Complement Factor I can bedetermined e.g. by detection of a reduction in the presence of C3b. Insome embodiments a polypeptide according to the present invention thatacts as a co-factor for Complement Factor I produces a smaller amount ofiC3b overall compared to an amount of iC3b produced by FH/FHL-1 viaComplement Factor I. In some embodiments a polypeptide according to thepresent invention that acts as a co-factor for Complement Factor Iincreases the ratio of C3dg to iC3b via Complement Factor I compared tothe ratio of C3dg to iC3b produced by FH/FHL-1 via Complement Factor l.For example, the polypeptide may not increase the overall amount ofiC3b, but may instead increase the amount of C3dg, C3f and/or C3d viaComplement Factor I.

In some embodiments a polypeptide according to the present invention iscapable of inhibiting or reducing complement activation. A polypeptidemay inhibit or reduce complement over-activation. The level ofcomplement activation/over-activation may be determined by the assaysdescribed herein, e.g. abnormal levels of complement components, or bytests/assays that are known by one skilled in the art, e.g. as describedin Shih and Murali Am. J. Hematol. 2015, 90: 1180-1186; Kirschfink andMollnes, Clin Diagn Lab Immunol. 2003, 10(6): 982-989; Nilsson andEkdahl, Clinical and Developmental Immunology, 2012, Article ID 962702;which are hereby incorporated by reference in their entirety.

In some embodiments, the polypeptide according to the present inventionpossesses the ability to diffuse through, i.e. pass through, Bruch’sMembrane (BrM), as determined by analysis in an appropriate assay forsaid property.

The ability of a given polypeptide to diffuse through BrM can beanalysed e.g. in vitro, e.g. as described in Clark et al J. Immunol(2014) 193, 4962-4970. Briefly, BrM can be isolated from donor eyes asdescribed in McHarg et al., J Vis Exp (2015) 1-7, and the macular areacan be mounted in an Ussing chamber. Once mounted, the 5 mm diametermacular area is the only barrier between two identical compartments.Both sides of BrM can be washed with PBS, and human serum can be diluted1:1 with PBS and added to the Ussing compartment on one side of the BrM(the sample chamber). The polypeptide to be analysed can be added to thesample chamber in PBS, and PBS alone can be added to the compartment onthe other side of the BrM (the diffusate chamber), and the Ussingchamber can be incubated at room temperature for 24 hours with gentlestirring in both the sample and diffusate chambers. Samples from eachchamber can subsequently be analysed for the presence of thepolypeptide, e.g. using antibody based detection methods such as ELISAanalysis or western blot. Detection of the polypeptide in the diffusatechamber indicates that the polypeptide is capable of diffusing throughBrM. Suitable positive and negative control proteins known to be ableto/not to be able to diffuse through BrM can be included in suchexperiments.

In some embodiments, a polypeptide according to the present inventiondisplays superior ability to diffuse through BrM than Complement FactorI. In some embodiments, a polypeptide according to the present inventiondisplays superior ability to diffuse through BrM than Complement FactorH. FH, consisting of 20 CCP domains, is a large molecule and does notpass through BrM. In some embodiments, a polypeptide according to thepresent invention displays similar ability to diffuse through BrM ascompared to the truncated Complement Factor H isoform FHL-1 (UniProt:P08603-2; SEQ ID NO:28). In some embodiments, a polypeptide according tothe present invention displays superior ability to diffuse through BrMas compared to Complement Factor H isoform FHL-1. In some embodiments, apolypeptide according to the present invention displays similar abilityto diffuse through BrM as compared to full length soluble CR1 (30 CCPdomains; SEQ ID NO:1 lacking SEQ ID NO:32 and 33). In some embodiments,a polypeptide according to the present invention displays superiorability to diffuse through BrM as compared to full length soluble CR1. Apolypeptide according to the present invention that is able to diffusethough BrM preferably remains functionally active, i.e. acts as acofactor for Complement Factor I, after diffusing though BrM.

A polypeptide of the present invention displaying superior ability todiffuse through BrM as compared to a given reference polypeptide can beidentified by analysing diffusion through BrM as described above. Thediffusion through BrM may be detected by measuring the rate of diffusionthrough to the diffusate chamber and/or detecting the proportion ofpolypeptide present in the diffusate chamber at the end of theexperiment. A polypeptide of the present invention displaying similarability to diffuse through BrM as compared to a given referencepolypeptide can be identified by analysing diffusion through BrM asdescribed above. A similar ability to diffuse through BrM may beindicated by detecting a rate of diffusion through to the diffusatechamber which is within 30%, e.g. within one of 25%, 20%, 15%, or 10% ofthe rate of diffusion for a reference polypeptide, and/or by detecting aproportion of the polypeptide of the present invention present in thediffusate chamber at the end of the experiment that is within 30%, e.g.within one of 25%, 20%, 15%, or 10% of the proportion of a referencepolypeptide present in the diffusate chamber.

As a result of the ability of the polypeptides to diffuse through theBrM, the polypeptides of the present invention are also able to diffuseaway from the site of C3b inactivation if/once they have performed theircofactor role with Fl. In other words, the polypeptides of the presentinvention may be capable of being present transiently at areas ofcomplement activation. This is advantageous because accumulation ofcomplement-related debris is undesirable, particularly in the context ofmacular degeneration where cellular debris accumulation can lead to theformation of drusen.

A polypeptide of the present invention may be capable of being expressedin a cell, e.g. a cell as described herein. A polypeptide of the presentinvention may be capable of being secreted by a cell, e.g. a cell asdescribed herein. In some embodiments the cell is an ocular cell, e.g.an RPE cell, as described herein.

Nucleic Acids, Cells, Compositions and Kits

The present invention provides a nucleic acid encoding a polypeptideaccording to the present invention. In some embodiments, the nucleicacid is purified or isolated, e.g. from other nucleic acid, ornaturally-occurring biological material. In some embodiments the nucleicacid(s) comprise or consist of DNA and/or RNA.

Provided herein are nucleic acid sequences that encode a polypeptidecomprising or consisting of SEQ ID NO:2, 3, 5, 6, 13, 14, 15, 30, 31,40, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53 or 54. The encodedpolypeptide may be produced with or without a leader sequence e.g. asecretory pathway sequence. The encoded polypeptide may be producedtogether with a leader sequence which is then subsequently removed fromsaid polypeptide.

In some embodiments, a nucleic acid according to the present inventioncomprises, or consists of, one or more of SEQ ID NO:35, 36, 37, 38, 39,41, 43, and/or 45, or equivalent nucleic acid sequences thereof whichwould be translated into the same respective polypeptides due to codondegeneracy.

The present invention also provides a vector comprising nucleic acidencoding a polypeptide according to the present invention.

The nucleotide sequence may be contained in a vector, e.g. an expressionvector. A “vector” as used herein is a nucleic acid molecule used as avehicle to transfer exogenous nucleic acid into a cell. The vector maybe a vector for expression of the nucleic acid in the cell. Such vectorsmay include a promoter sequence operably linked to the nucleotidesequence encoding the sequence to be expressed. A vector may alsoinclude a termination codon and expression enhancers. A vector mayinclude regulatory elements, such as a polyadenylation site. Anysuitable vectors, promoters, enhancers and termination codons known inthe art may be used to express a peptide or polypeptide from a vectoraccording to the invention. Nucleic acid sequences described herein maybe codon optimised for optimised expression in a desired cell ororganism.

The term “operably linked” may include the situation where a selectednucleic acid sequence and regulatory nucleic acid sequence (e.g.promoter and/or enhancer) are covalently linked in such a way as toplace the expression of nucleic acid sequence under the influence orcontrol of the regulatory sequence (thereby forming an expressioncassette). Thus a regulatory sequence is operably linked to the selectednucleic acid sequence if the regulatory sequence is capable of effectingtranscription of the nucleic acid sequence. The resulting transcript(s)may then be translated into a desired peptide(s)/polypeptide(s).

The nucleic acid and/or vector according to the present invention ispreferably provided for introduction into a cell, e.g. a human cell.Suitable vectors include plasmids, binary vectors, DNA vectors, mRNAvectors, viral vectors (e.g. gammaretroviral vectors (e.g. murineLeukemia virus (MLV)-derived vectors), lentiviral vectors, retroviralvectors, adenovirus vectors, adeno-associated virus (AAV) vectors,vaccinia virus vectors and herpesvirus vectors, e.g. Herpes SimplexVirus vectors), transposon-based vectors, and artificial chromosomes(e.g. yeast artificial chromosomes), e.g. as described in Maus et al.,Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines2016 4, 9, which are both hereby incorporated by reference in itsentirety. In some embodiments, the lentiviral vector may be pELNS, ormay be derived from pELNS. In some embodiments, the vector may be avector encoding CRISPR/Cas9. In some embodiments, the adeno-associatedvirus (AAV) vector is selected from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or 11, or hybrids and/or mutants thereof. In some embodiments, theAAV vector is an AAV serotype 2 (AAV-2) vector, or a hybrid and/ormutant thereof. Viral and non-viral delivery systems for introducinggenetic material into cells are reviewed, for example, in Nayerossadatet al., Adv Biomed Res. 2012; 1: 27; MacLaren et al. Ophthalmology.2016, 123(10 Suppl): S98-S106; Petit and Punzo, Discov Med. 2016,22(121): 221-229; Aguirre, Invest Ophthalmol Vis Sci. 2017, 58(12):5399-5411; Lundstrom, Diseases. 2018, 6(2): 42; which are herebyincorporated by reference in their entirety. Any suitable nucleotide orvector delivery method can be used in the context of the presentinvention.

In some embodiments the expression of a nucleic acid or a nucleic acidcontained in a vector, according to the present invention, is driven bya promoter that drives expression in a specific retinal cell type, e.g.rods, cones, RPE, or ganglion cells, as described for example in BeltranWA, et al. Gene Ther. 2010; 17:1162-74 and Boye SE, et al. Hum GeneTher. 2012; 23:1101-15, which are hereby incorporated by reference intheir entirety.

In some embodiments, the expression of a nucleic acid or a nucleic acidcontained in a vector, according to the present invention, is driven bya promoter that drives expression of that nucleic acid in retinalpigment epithelial (RPE) cells. In some embodiments, the promoter is anRPE65 or VMD2 promoter, or modified version thereof. In some embodimentsthe promoter is a chicken β actin promoter.

In some embodiments, the vector may be a eukaryotic vector, e.g. avector comprising the elements necessary for expression of protein fromthe vector in a eukaryotic cell. In some embodiments, the vector may bea mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40promoter to drive protein expression.

In some embodiments the vector comprises an inducible promoter, i.e.gene expression is activated by the promoter only in the presence orabsence of a particular molecule. Suitable inducible promoters will beknown to the skilled person. Examples of inducible promoters aredescribed in e.g. Le at al. Invest Ophthalmol Vis Sci. 2008, 49(3):1248-1253 and McGee Sanftner et al. Mol Ther. 2001. 3(5): 688-696; whichare hereby incorporated by reference in their entirety.

In some embodiments, the nucleic acid comprises, or consists of, anucleic acid sequence encoding a polypeptide having an amino acidsequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO:2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 30, 31, 40, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, or 54,or any combination of sequences A, B, and/or C as described hereinabove.

The present invention also provides a cell comprising or expressing apolypeptide according to the present invention. Also provided is a cellcomprising or expressing a nucleic acid or vector according to theinvention. The cell comprising or expressing polypeptide, nucleic acidor vector according to the present invention may secrete a polypeptideaccording to the present invention. That is, expression of thepolypeptide, nucleic acid or vector by the cell may result in thesoluble production of a polypeptide according of the present inventionfrom the cell.

The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal maybe a human, or a non-human mammal (e.g. rabbit, guinea pig, rat, mouseor other rodent (including any animal in the order Rodentia), cat, dog,pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animalin the order Bos), horse (including any animal in the order Equidae),donkey, and non-human primate). In some embodiments, the cell may befrom, or may have been obtained from, a human subject.

In some embodiments, the cell is a cell of the eye. In some embodiments,the cell is a cell of the neurosensory retina, retinal pigmentepithelium (RPE), choroid or macula. In some embodiments, the cell is aretinal cell. In some embodiments, the cell is a retinal pigmentepithelial cell. In some embodiments, the cell is a human retinalpigment epithelial cell (RPE). In some embodiments the cell is aphotoreceptor cell.

The present invention also provides a method for producing a cellcomprising a nucleic acid or vector according to the present invention,comprising introducing a nucleic acid or vector according to the presentinvention into a cell. In some embodiments, introducing an isolatednucleic acid(s) or vector(s) according to the invention into a cellcomprises transformation, transfection, electroporation or transduction(e.g. retroviral transduction). Methods for producing a cell accordingto the present invention may be performed according to methods known tothe skilled person for the production of cells comprising nucleicacid/vectors.

The present invention also provides a method for producing a cellcomprising or expressing a polypeptide according to the presentinvention, comprising introducing a nucleic acid or vector according tothe present invention into a cell. In some embodiments, the methodsadditionally comprise culturing the cell under conditions suitable forexpression of the nucleic acid or vector by the cell. In someembodiments, the methods are performed in vitro or ex vivo. In someembodiments, the methods are performed in vivo.

The present invention also provides cells obtained or obtainable by themethods according to the present invention.

The present invention also provides compositions comprising apolypeptide, nucleic acid, vector, or cell according to the presentinvention.

Polypeptides, nucleic acids, vectors and cells according to the presentinvention may be formulated as pharmaceutical compositions for clinicaluse and may comprise a pharmaceutically acceptable carrier, diluent,excipient or adjuvant.

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions, such methods ofproduction may comprise one or more steps selected from: isolating apolypeptide, cell, nucleic acid or vector as described herein; and/ormixing a polypeptide, cell, nucleic acid or vector as described hereinwith a pharmaceutically acceptable carrier, adjuvant, excipient ordiluent.

A kit of parts is also provided. In some embodiments the kit may have atleast one container having a predetermined quantity of a polypeptide,nucleic acid, vector, cell, and/or composition according to the presentinvention.

The kit may provide the polypeptide, nucleic acid, vector, cell orcomposition together with instructions for administration to a subjectin order to treat a specified disease/condition. The polypeptide,nucleic acid, vector, cell or composition may be formulated so as to besuitable for injection or infusion. In some embodiments, thepolypeptide, nucleic acid, vector, cell or composition may be formulatedso as to be suitable for intravenous, intraocular, sub-retinal,suprachoroidal or intraconjunctival injection, administration as an eyedrop (i.e. ophthalmic administration), or oral administration.

In some embodiments the kit may comprise materials for producing a cellaccording to the present invention. For example, the kit may comprisematerials for modifying a cell to express or comprise a polypeptide,nucleic acid or vector according to the present invention, or materialsfor introducing into a cell the nucleic acid or vector according to thepresent invention.

In some embodiments the kit may further comprise at least one containerhaving a predetermined quantity of another therapeutic agent (e.g. atherapeutic agent for the treatment of AMD). In such embodiments, thekit may also comprise a second medicament or pharmaceutical compositionsuch that the two medicaments or pharmaceutical compositions may beadministered simultaneously or separately such that they provide acombined treatment for the specific disease or condition. In someembodiments, the second medicament or pharmaceutical compositioncomprises Complement Factor I.

Producing Polypeptides

The present invention also provides a method for producing a polypeptideaccording to the present invention, the method comprising introducinginto a cell a nucleic acid or vector according to the present invention,and culturing the cell under conditions suitable for expression of thepolypeptide. The polypeptide may be a fusion protein. The polypeptidemay be subsequently isolated and/or substantially purified.

Polypeptides according to the present invention may be preparedaccording to methods for the production of polypeptides known to theskilled person.

Polypeptides may be prepared by chemical synthesis, e.g. liquid or solidphase synthesis. For example, peptides/polypeptides can by synthesisedusing the methods described in, for example, Chandrudu et al., Molecules(2013), 18: 4373-4388, which is hereby incorporated by reference in itsentirety.

Alternatively, polypeptides may be produced by recombinant expression.Molecular biology techniques suitable for recombinant production ofpolypeptides are well known in the art, such as those set out in Greenand Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), ColdSpring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146both of which are hereby incorporated by reference in their entirety.

For recombinant production according to the invention, any cell suitablefor the expression of polypeptides may be used. The cell may be aprokaryote or eukaryote. In some embodiments the cell is a prokaryoticcell, such as a cell of archaea or bacteria. In some embodiments thebacteria may be Gram-negative bacteria such as bacteria of the familyEnterobacteriaceae, for example Escherichia coli. In some embodiments,the cell is a eukaryotic cell such as a yeast cell, a plant cell, insectcell or a mammalian cell, e.g. CHO, HEK (e.g. HEK293), HeLa or COScells.

In some cases the cell is not a prokaryotic cell because someprokaryotic cells do not allow for the same folding orpost-translational modifications as eukaryotic cells. In addition, veryhigh expression levels are possible in eukaryotes and proteins can beeasier to purify from eukaryotes using appropriate tags. Specificplasmids may also be utilised which enhance secretion of the proteininto the media.

In some embodiments polypeptides may be prepared by cell-free-proteinsynthesis (CFPS), e.g. according using a system described in Zemella etal. Chembiochem (2015) 16(17): 2420-2431, which is hereby incorporatedby reference in its entirety.

Production may involve culture or fermentation of a eukaryotic cellmodified to express the polypeptide(s) of interest. The culture orfermentation may be performed in a bioreactor provided with anappropriate supply of nutrients, air/oxygen and/or growth factors.Secreted proteins can be collected by partitioning culturemedia/fermentation broth from the cells, extracting the protein content,and separating individual proteins to isolate secreted polypeptide(s).Culture, fermentation and separation techniques are well known to thoseof skill in the art, and are described, for example, in Green andSambrook, Molecular Cloning: A Laboratory Manual (4th Edition;incorporated by reference herein above).

Bioreactors include one or more vessels in which cells may be cultured.Culture in the bioreactor may occur continuously, with a continuous flowof reactants into, and a continuous flow of cultured cells from, thereactor. Alternatively, the culture may occur in batches. The bioreactormonitors and controls environmental conditions such as pH, oxygen, flowrates into and out of, and agitation within the vessel such that optimumconditions are provided for the cells being cultured.

Following culturing the cells that express the antigen-bindingmolecule/polypeptide(s), the polypeptide(s) of interest may be isolated.Any suitable method for separating proteins from cells known in the artmay be used. In order to isolate the polypeptide it may be necessary toseparate the cells from nutrient medium. If the polypeptide(s) aresecreted from the cells, the cells may be separated by centrifugationfrom the culture media that contains the secreted polypeptide(s) ofinterest. If the polypeptide(s) of interest collect within the cell,protein isolation may comprise centrifugation to separate cells fromcell culture medium, treatment of the cell pellet with a lysis buffer,and cell disruption e.g. by sonication, rapid freeze-thaw or osmoticlysis.

It may then be desirable to isolate the polypeptide(s) of interest fromthe supernatant or culture medium, which may contain other protein andnon-protein components. A common approach to separating proteincomponents from a supernatant or culture medium is by precipitation.Proteins of different solubilities are precipitated at differentconcentrations of precipitating agent such as ammonium sulfate. Forexample, at low concentrations of precipitating agent, water solubleproteins are extracted. Thus, by adding different increasingconcentrations of precipitating agent, proteins of differentsolubilities may be distinguished. Dialysis may be subsequently used toremove ammonium sulfate from the separated proteins.

Other methods for distinguishing different proteins are known in theart, for example ion exchange chromatography and size chromatography.The polypeptide may also be affinity-purified using an appropriatebinding partner for a molecular tag on the polypeptide (e.g. a His,FLAG, Myc, GST, MBP, HA, E, or Biotin tag). These may be used as analternative to precipitation, or may be performed subsequently toprecipitation.

In some cases it may further be desired to process the polypeptide, e.g.to remove a sequence of amino acids, molecular tag, moiety, etc.

In some embodiments, treatment is with an appropriate endopeptidase forthe cleavage and removal of an amino acid sequence.

In some embodiments, treatment is with an enzyme to remove the moiety ofinterest. In some embodiments, the polypeptide is treated to removeglycans (i.e. the polypeptide is degylcosylated), e.g. by treatment witha glycosidase such as with a Peptide:N-glycosidase (PNGase).

Once the polypeptide(s) of interest have been isolated from culture itmay be desired or necessary to concentrate the polypeptide(s). A numberof methods for concentrating proteins are known in the art, such asultrafiltration or lyophilisation.

In some embodiments, the production of the polypeptide occurs in vivo,e.g. after introduction to the host of a cell comprising a nucleic acidor vector encoding a polypeptide of the present invention, or followingintroduction into a cell of the host of a nucleic acid or vectorencoding a polypeptide of the present invention. In such embodiments,the polypeptide is transcribed, translated and post-translationallyprocessed to the mature polypeptide. In some embodiments, thepolypeptide is produced in situ at the desired location in the host. Insome embodiments, the desired location is the eye, e.g. in a cell of theretina, choroid, retinal pigment epithelium (RPE) or macula. In someembodiments, the desired location is at or in a retinal cell. In someembodiments, the desired location is at or in a RPE cell.

Therapeutic Applications

Any of the polypeptides, nucleic acids, vectors, cells andpharmaceutical compositions according to the present invention find usein therapeutic and prophylactic methods.

The present invention provides a polypeptide, nucleic acid, vector,cell, or pharmaceutical composition according to the present invention,for use in a method of medical treatment or prophylaxis. The presentinvention also provides the use of a polypeptide, nucleic acid, vector,cell or pharmaceutical composition according to the present invention inthe manufacture of a medicament for treating or preventing a disease orcondition. The present invention also provides a method of treating orpreventing a disease or condition, comprising administering to a subjecta therapeutically or prophylactically effective amount of a polypeptide,nucleic acid, vector, cell or pharmaceutical composition according tothe present invention.

In particular, the polypeptides, nucleic acids, vectors, cells andpharmaceutical compositions according to the present invention find useto treat or prevent diseases/conditions associated with complementdysregulation, in particular overactive complement response. In someembodiments, the overactive complement response is linked to thepresence of C3b. In some embodiments the disease/condition to be treatedor prevented is a complement-related disease. In some embodiments thedisease/condition to be treated or prevented is pathologicallyassociated with complement activation. In some embodiments thedisease/condition to be treated or prevented is pathologicallyassociated with complement over-activation. In some embodiments thedisease/condition to be treated or prevented is driven by complementactivation or over-activation. In some embodiments the disease/conditionis complement activation or over-activation.

The polypeptides, nucleic acids, vectors, cells and pharmaceuticalcompositions find use to treat or prevent diseases/conditions whichwould benefit from one or more of: a reduction in the level or activityof C3bBb-type C3 convertase, C3bBb3b-type C5 convertase or C4b2a3b-typeC5 convertase; a reduction in the level of C3b, C5b or C5a; an increasein the level of iC3b, C3f, C3dg or C3d; or a reduction in the level oractivity of iC3b and an increase in the level of C3f, C3dg or C3d.

‘Treatment’ may, for example, be reduction in the development orprogression of a disease/condition, alleviation of the symptoms of adisease/condition or reduction in the pathology of a disease/condition.Treatment or alleviation of a disease/condition may be effective toprevent progression of the disease/condition, e.g. to prevent worseningof the condition or to slow the rate of development. In some embodimentstreatment or alleviation may lead to an improvement in thedisease/condition, e.g. a reduction in the symptoms of thedisease/condition or reduction in some other correlate of theseverity/activity of the disease/condition. Prevention/prophylaxis of adisease/condition may refer to prevention of a worsening of thecondition or prevention of the development of the disease/condition,e.g. preventing an early stage disease/condition developing to a later,chronic, stage.

In some embodiments, the disease or condition to be treated or preventedmay be a disease/condition associated with C3b or a C3b-containingcomplex, an activity/response associated with C3b or a C3b-containingcomplex, or a product of an activity/response associated with C3b or aC3b-containing complex. That is, in some embodiments, the disease orcondition to be treated or prevented is a disease/condition in whichC3b, a C3b-containing complex, an activity/response associated with C3bor a C3b-containing complex, or the product of said activity/response ispathologically implicated. In some embodiments, the disease/conditionmay be associated with an increased level of C3b or a C3b-containingcomplex, an increased level of an activity/response associated with C3bor a C3b-containing complex, or increased level of a product of anactivity/response associated with C3b or a C3b-containing complex ascompared to the control state.

The treatment may be aimed at reducing the level of C3b or aC3b-containing complex, an activity/response associated with C3b or aC3b-containing complex, or a product of an activity/response associatedwith C3b or a C3b-containing complex. In some embodiments, the treatmentis aimed at: reducing the level or activity of C3bBb-type C3 convertase,C3bBb3b-type C5 convertase or C4b2a3b-type C5 convertase; reducing thelevel of C3b, C5b or C5a; increasing the level of iC3b, C3f, C3dg orC3d, or reducing the level of iC3b and increasing the level of C3f, C3dgor C3d.

Administration of the polypeptides, nucleic acids, vectors, cells andcompositions of the present invention may cause a reduction in the levelof C3b or a C3b-containing complex, an activity/response associated withC3b or a C3b-containing complex, or a product of an activity/responseassociated with C3b or a C3b-containing complex through cleavage of C3b.

In some embodiments, the treatment may be aimed at reducing the level ofC3b or a C3b-containing complex, an activity/response associated withC3b or a C3b-containing complex, or a product of an activity/responseassociated with C3b or a C3b-containing complex in a subject, e.g. at aparticular location, in a particular organ, tissue, structure or celltype. In some embodiments, the treatment may be aimed at reducing thelevel of C3b or a C3b-containing complex, an activity/responseassociated with C3b or a C3b-containing complex, or a product of anactivity/response associated with C3b or a C3b-containing complex in theeye, e.g. in the retina, choroid, RPE, macula and/or at the BrM/RPEinterface.

In some embodiments, the treatment may comprise modifying a cell orpopulation of cells to comprise/express a polypeptide, nucleic acid orvector of the present invention. In some embodiments, the treatment maycomprise modification of the cell/population in vivo, for in situproduction of the polypeptide of the invention. In some embodiments thecell/population of cells is/are an ocular cell/cells. In someembodiments, the cell/population of cells is a RPE cell and/orpopulation of RPE cells. In some embodiments the cell/population ofcells is a photoreceptor cell and/or population of photoreceptor cells.

In some embodiments, the present invention provides a nucleic acid orvector of the present invention for use in gene therapy. In someembodiments, the treatment comprises administering the nucleic acidand/or vector to a subject. In some embodiments, the treatment comprisesintroducing the nucleic acid and/or vector into a cell of a subject,using techniques described herein or well known in the art, see e.g.MacLaren et al. Ophthalmology. 2016, 123(10 Suppl): S98-S106; Aguirre,Invest Ophthalmol Vis Sci. 2017, 58(12): 5399-5411; Lundstrom, Diseases.2018, 6(2): 42; which are hereby incorporated by reference in theirentirety. In some embodiments the cell is an ocular cell or cells. Insome embodiments, the cell is an RPE cell or cells. In some embodimentsthe cell is a photoreceptor cell or cells.

In some embodiments, the treatment may comprise administering to asubject a cell or population of cells modified to comprise/express apolypeptide, nucleic acid or vector of the present invention. In someembodiments, the treatment may comprise modification of thecell/population ex vivo or in vitro.

In some embodiments, the treatment is aimed at providing the subjectwith a cell or population of cells which produce and/or will produce thepolypeptide of the invention, e.g. by administering a cell according tothe present invention, or by generating a cell according to the presentinvention.

In some embodiments, the cell refered to herein is a cell of the eyei.e. an ocular cell. In some embodiments, the cell is a cell of theretina, choroid, retinal pigment epithelium (RPE) or macula. In someembodiments, the cell is a retinal cell. In some embodiments, the cellis an RPE cell. In some embodiments the cell is a photoreceptor cell.

The present invention provides a method of treating or preventing adisease or condition in a subject, the method comprising modifying atleast one cell to express or comprise a polypeptide, nucleic acid orvector according to the present invention. In some embodiments the atleast one cell is an ocular cell. In some embodiments, the at least onecell is an RPE cell.

The at least one cell modified according to the present invention can bemodified according to methods well known to the skilled person. Themodification may comprise nucleic acid transfer for permanent ortransient expression of the transferred nucleic acid. Any suitablegenetic engineering platform may be used to modify a cell according tothe present invention. Suitable methods for modifying a cell include theuse of genetic engineering platforms such as gammaretroviral vectors,lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV)vectors, DNA transfection, transposon-based gene delivery and RNAtransfection, for example as described in Maus et al., Annu Rev Immunol(2014) 32:189-225, incorporated by reference hereinabove.

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient. A subject may have beendiagnosed with a disease or condition requiring treatment, or besuspected of having such a disease or condition.

The subject to be treated may display an elevated level of C3b or aC3b-containing complex, an activity/response associated with C3b or aC3b-containing complex, or a product of an activity/response associatedwith C3b or a C3b-containing complex, e.g. as determined by analysis ofthe subject, or a sample (e.g. a cell, tissue, blood sample) obtainedfrom the subject, using an appropriate assay.

The subject may have an increased level of expression or activity of apositive regulator/effector of C3b or a C3b-containing complex or of anactivity/response associated with C3b or a C3b-containing complex, ormay have an increased level of expression or activity of a product of anactivity/response associated with C3b or a C3b-containing complex. Thesubject may have an increased level of an activity upregulated by C3b ora C3b-containing complex.

The subject may have a reduced level of expression or activity of anegative regulator of C3b or a C3b-containing complex or of anactivity/response associated with C3b or a C3b-containing complex, ormay have a reduced level of expression or activity a factordownregulated by C3b or a C3b-containing complex. The subject may have areduced level of an activity downregulated by C3b or a C3b-containingcomplex.

The increase/reduction may be relative to the level ofexpression/activity in the absence of the relevant disease/condition,e.g. the level of expression/activity in a healthy control subject orsample obtained from a healthy control subject.

In some embodiments, the subject may be at risk ofdeveloping/contracting a disease or condition. In some embodiments, thesubject may possess one or more predisposing factors increasing risk ofdeveloping/contracting a disease or condition.

In some embodiments, the subject may possess one or more risk factorsfor Age-related Macular Degeneration (AMD). In some embodiments, thesubject may possess one or more of AMD-associated genetic variants.AMD-associated genetic variants are described e.g. in Clark et al., JClin Med (2015) 4(1):18-31, which is hereby incorporated by reference inits entirety. In some embodiments, the subject may possess one or moreof the following AMD-associated genetic variants (or a variant having LD= r² ≥ 0.8 with such variant): Y402H in CFH (i.e. rs1061170^(C)),rs1410996^(C), 162V in CFH, R53C in CFH, D90G in CFH, R1210C in CFH, orrs6685931^(T)in CFHR4.

In some embodiments the subject may possess one or more risk factors forearly-onset macular degeneration (EOMD). EOMD is thought to be caused bymonogenic inheritance of rare variants of the CFH gene (see e.g. Boon CJet al. Am J Hum Genet 2008; 82(2):516-23; van de Ven JP, et al. ArchOphthalmol 2012;130(8):1038-47; Yu Y et al. Hum Mol Genet 2014;23(19):5283-93; Duvvari MR, et al. Mol Vis 2015; 21:285-92; Hughes AE,et al. Acta Ophthalmol 2016; 94(3):e247-8; Wagner et al. Sci Rep2016;6:31531). In some embodiments, the subject may possess one or moreof EOMD-associated genetic variants. EOMD-associated genetic variantsare described in e.g. Servais A et al. Kidney Int, 2012; 82(4):454-64and Dragon-Durey MA, et al. J Am Soc Nephrol 2004; 15(3):787-95; whichare hereby incorporated by reference in their entirety. In someembodiments, the subject may possess one or more of the followingEOMD-associated genetic variants: CFH c.1243del, p.(Ala415Profs*39) het;CFH c.350+1G>T het; CFH c.619+1G>A het; CFH c.380G>A, p.(Arg127His); CFHc.694C>T, p.(Arg232Ter); or CFH c.1291T>A, p.(Cys431Ser).

In some embodiments, the subject is selected for therapeutic orprophylactic treatment with the polypeptide, nucleic acid, vector, cellor composition of the present invention based on their being determinedto possess one or more risk factors for AMD and/or EOMD, e.g. one ormore AMD/EOMD-associated genetic variants. In some embodiments, thesubject has been determined to have one or more such risk factors. Insome embodiments, the methods of the present invention involvingdetermining whether a subject possesses one or more such risk factors.

In some embodiments, the disease or condition to be treated or preventedmay be an ocular disease/condition. In some embodiments, the disease orcondition to be treated or prevented is a complement-related oculardisease. In some embodiments, the disease or condition to be treated orprevented is macular degeneration. In some embodiments, the disease orcondition to be treated or prevented is age-related macular degeneration(AMD). AMD is commonly-defined as causing vision loss in subjects age 50and older.

In some embodiments, the disease or condition to be treated or preventedis selected from age-related macular degeneration (AMD), early AMD,intermediate AMD, late AMD, geographic atrophy (‘dry’ (i.e.non-exudative) AMD), ‘wet’ (neovascular or exudative) AMD, choroidalneovascularisation (CNV), glaucoma, autoimmune uveitis, and diabeticretinopathy. In some embodiments, the disease or condition to be treatedor prevented is AMD. In some embodiments, the disease or condition to betreated or prevented is geographic atrophy (‘dry’ AMD). In someembodiments, the disease or condition to be treated or prevented is‘wet’ AMD. In some embodiments the disease or condition to be treated orprevented is a combination of the diseases/conditions above, e.g. ‘dry’and ‘wet’ AMD. In some embodiments the disease or condition to betreated or prevented is not ‘wet’ AMD or choroidal neovascularisation.In some embodiments a subject to be treated is age 50 or older, i.e. isat least 50 years old.

As used herein “early AMD” refers to a stage of AMD characterised by thepresence of medium-sized drusen, commonly having a width of up to ~200µm, within the Bruch’s membrane adjacent to the RPE layer. Subjects withearly AMD typically do not present significant vision loss. As usedherein “intermediate AMD” refers to a stage of AMD characterised bylarge drusen and/or pigment changes in the retina. Intermediate AMD maybe accompanied by some vision loss. As used herein “late AMD” refers toa stage of AMD characterised by the presence of drusen and vision lossdue to damage to the macula. In all stages of AMD, ‘reticularpseudodrusen’ (RPD) or ‘reticular drusen’ may be present, referring tothe accumulation of extracellular material in the subretinal spacebetween the neurosensory retina and RPE. “Late AMD” encompasses ‘dry’and ‘wet’ AMD. In ‘dry’ AMD (also known as geographic atrophy), there isa gradual breakdown of the light-sensitive cells in the macula thatconvey visual information to the brain and of the supporting tissuebeneath the macula. In ‘wet’ AMD (also known as choroidalneovascularization and exudative AMD), abnormal blood vessels growunderneath and into the retina. These vessels can leak fluid and bloodwhich can lead to swelling and damage of the macula and subsequent scarformation. The damage may be rapid and severe.

In some embodiments the disease or condition to be treated or preventedis early-onset macular degeneration (EOMD). As used herein “EOMD” refersto a phenotypically severe sub-type of macular degeneration thatdemonstrates a much earlier age of onset than classical AMD and resultsin many more years of substantial visual loss. The EOMD subset isdescribed in e.g. Boon CJ et al. Am J Hum Genet 2008; 82(2):516-23 andvan de Ven JP, et al. Arch Ophthalmol 2012;130(8):1038-47. In someembodiments a subject to be treated is age 49 or younger. In someembodiments a subject to be treated is between ages 15 and 49, i.e. isbetween 15 and 49 years old.

In some embodiments, the disease or condition to be treated or preventedis a disease/condition driven by complement over-activation. In someembodiments, the disease or condition to be treated or prevented may beselected from atypical Haemolytic Uremic Syndrome (aHUS),Membranoproliferative Glomerulonephritis Type II (MPGN II), sepsis, andParoxysmal nocturnal hemoglobinuria (PNH).

Methods of medical treatment may also involve in vivo, ex vivo, andadoptive immunotherapies, including those using autologous and/orheterologous cells or immortalized cell lines.

Administration of a polypeptide described herein is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofthe disease being treated. Prescription of treatment, e.g. decisions ondosage etc., is within the responsibility of general practitioners andother medical doctors, and typically takes account of the condition tobe treated, the condition of the individual patient, the site ofdelivery, the method of administration and other factors known topractitioners. Examples of the techniques and protocols mentioned abovecan be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000,pub. Lippincott, Williams & Wilkins.

Polypeptides, nucleic acids, vectors and cells according to the presentinvention may be formulated as pharmaceutical compositions ormedicaments for clinical use and may comprise a pharmaceuticallyacceptable carrier, diluent, excipient or adjuvant. The composition maybe formulated for topical, parenteral, systemic, intracavitary,intravenous, intra-arterial, intramuscular, intrathecal, intraocular,intraconjunctival, subretinal, suprachoroidal, subcutaneous,intradermal, intrathecal, oral or transdermal routes of administrationwhich may include injection or infusion, or administration as an eyedrop (i.e. ophthalmic administration). Suitable formulations maycomprise the polypeptide, nucleic acid, vector, or cell in a sterile orisotonic medium. Medicaments and pharmaceutical compositions may beformulated in fluid, including gel, form. Fluid formulations may beformulated for administration by injection or infusion (e.g. viacatheter) to a selected organ or region of the human or animal body. Insome embodiments the polypeptides, nucleic acids, vector, cells andcompositions of the invention are formulated for intravitreal routes ofadministration e.g. by intravitreal injection. In some embodimentspolypeptides, nucleic acids, vector, cells and compositions of theinvention are formulated for submacular delivery i.e. placing thetherapeutic molecules in direct contact with the target cell layers.

The particular mode and/or site of administration may be selected inaccordance with the location where the C3b inactivation is desired. Insome embodiments, the polypeptides, nucleic acids, vectors, orpharmaceutical compositions of the present invention are formulated foradministration and/or administered into the subretinal space between thephotoreceptor cells and the retinal pigment epithelium (RPE) in the eye.In some embodiments, the polypeptides, nucleic acids, vectors, orpharmaceutical compositions of the present invention are formulated foradministration and/or administered into the retinal pigment epithelium(RPE).

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions, such methods ofproduction may comprise one or more steps selected from: isolating apolypeptide, nucleic acid, vector, or cell as described herein; and/ormixing a polypeptide, nucleic acid, vector, or cell as described hereinwith a pharmaceutically acceptable carrier, adjuvant, excipient ordiluent.

For example, a further aspect of the present invention relates to amethod of formulating or producing a medicament or pharmaceuticalcomposition for use in a method of medical treatment, the methodcomprising formulating a pharmaceutical composition or medicament bymixing polypeptide, nucleic acid, vector, or cell as described hereinwith a pharmaceutically acceptable carrier, adjuvant, excipient ordiluent.

Administration may be alone or in combination with other treatments(e.g. other therapeutic or prophylactic intervention), eithersimultaneously or sequentially dependent upon the condition to betreated. The polypeptide, nucleic acid, vector, cell or compositionaccording to the present invention and a therapeutic agent may beadministered simultaneously or sequentially.

Simultaneous administration refers to administration of the polypeptide,nucleic acid, vector, cell or composition and therapeutic agenttogether, for example as a pharmaceutical composition containing bothagents (combined preparation), or immediately after each other andoptionally via the same route of administration, e.g. to the sametissue, artery, vein or other blood vessel. Sequential administrationrefers to administration of one of the polypeptide, nucleic acid,vector, cell or composition or therapeutic agent followed after a giventime interval by separate administration of the other agent. It is notrequired that the two agents are administered by the same route,although this is the case in some embodiments. The time interval may beany time interval. In some embodiments, the polypeptide, nucleic acid,vector, cell or composition and therapeutic agent are administeredseparately, simultaneously or sequentially to the eye.

In some embodiments, the other treatment/therapeutic agent is atherapeutically effective amount of Complement Factor l. In someembodiments, Complement Factor I is administered to the subjectsimultaneously or sequentially with administration of a polypeptide,nucleic acid, vector, cell, or pharmaceutical composition according tothe present invention. In some embodiments, the treatment may comprisemodifying a cell or population of cells in vitro, ex vivo or in vivo toexpress and/or secrete Complement Factor I. The cell or population ofcells may be the same cell or population of cells as a cell orpopulation of cells modified to comprise/express a polypeptide, nucleicacid or vector according to the present invention, for example thetreatment may comprise modifying a cell or population of cells in vitro,ex vivo or in vivo to express and/or secrete a polypeptide, nucleic acidor vector according to the present invention, and Complement Factor I.In some embodiments, Complement Factor I is administered to a subject,wherein the subject comprises a cell or population of cells modified tocomprise/express a polypeptide, nucleic acid or vector of the presentinvention. In some embodiments, Complement Factor I is administered to asubject wherein the subject has expressed in situ or is expressing insitu a polypeptide, nucleic acid or vector of the present invention.

Complement Factor I, or a composition comprising Complement Factor I,may be formulated for topical, parenteral, systemic, intracavitary,intravenous, intravitreal, intra-arterial, intramuscular, intrathecal,intraocular, intraconjunctival, subretinal, suprachoroidal,subcutaneous, intradermal, intrathecal, oral or transdermal routes ofadministration which may include injection or infusion, oradministration as an eye drop (i.e. ophthalmic administration). Suitableformulations may comprise a sterile or isotonic medium. Medicaments andpharmaceutical compositions may be formulated in fluid, including gel,form. Fluid formulations may be formulated for administration byinjection or infusion (e.g. via catheter) to a selected organ or regionof the human or animal body.

In some embodiments, the other treatment/therapeutic agent is atherapeutically effective amount of an anti-VEGF therapy (e.g.ranibizumab (Lucentis; Genentech/Novartis), bevacizumab (off labelAvastin; Genentech), aflibercept (Eylea/VEGF Trap-Eye;Regeneron/Bayer)), pegaptanib (Macugen®), laser photocoagulation, orphotodynamic therapy (PDT) e.g. with Visudyne™ (verteporfin).

Multiple doses of the polypeptide, nucleic acid, vector, cell orcomposition may be provided. One or more, or each, of the doses may beaccompanied by simultaneous or sequential administration of anothertherapeutic agent.

Multiple doses may be separated by a predetermined time interval, whichmay be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may begiven once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

A polypeptide, nucleic acid, vector or composition according to thepresent invention may be formulated in a sustained release deliverysystem, in order to release the polypeptide, nucleic acid, vector orcomposition at a predetermined rate. Sustained release delivery systemsmay maintain a constant drug/therapeutic concentration for a specifiedperiod of time. In some embodiments, a polypeptide, nucleic acid, vectoror composition according to the present invention is formulated in aliposome, gel, implant, device, or drug-polymer conjugate e.g. hydrogel.

Genetic Factors in Age-Related Macular Degeneration (AMD)

Complement Factor H (encoded by the CFH gene) is another co-factor forComplement Factor I. Complement Factor H structure and function isreviewed e.g. in Wu et al., Nat Immunol (2009) 10(7): 728-733, which ishereby incorporated by reference in its entirety. Human ComplementFactor H (UniProt: P08603; SEQ ID NO:29) has a 1,233 amino acid sequence(including an N-terminal, 18 amino acid signal peptide), and comprises20 complement control protein (CCP) domains. The first four CCP domains(i.e. CCP1 to CCP4) of Complement Factor H are necessary for ComplementFactor I co-factor activity for cleavage of C3b to iC3b. CCPs 19 to 20have also been shown to engage with C3b and C3d (Morgan et al., NatStruct Mol Biol (2011) 18(4): 463-470), whilst CCP7 and CCPs 19 to 20bind to glycosaminoglycans (GAGs) and sialic acid, and are involved indiscrimination between self and non-self (Schmidt et al., J Immunol(2008) 181(4): 2610-2619; Kajander et al., PNAS (2011) 108(7):2897-2902).

One of the major SNPs associated with genetic risk of developing AMD isfound in the CFH gene and leads to the Y402H polymorphism in ComplementFactor H (see e.g. Haines et al., Science (2005) 308:419-21), and it’salternative splice variant factor H-like protein 1 (FHL-1). Around 30%of individuals of white European heritage have at least one copy of thispolymorphism, whilst being a heterozygote increases the risk of AMD by~3-fold (Sofat et al., Int J Epidemiol (2012) 41:250-262). The Y402Hpolymorphism, which manifests in the seventh complement control protein(CCP) domain, reduces the binding of FH/FHL-1 to BrM, leading toperturbations in the binding of these blood-borne complement regulatorsand dampened complement regulation on this surface (Clark et al., J BiolChem (2010) 285:30192-202).

The binding of FH/FHL-1 to BrM is mediated by sulphated sugars includingthe glycosaminoglycans (GAGs) heparan sulphate (HS) and dermatansulphate (DS). The family of GAG sequences found in BrM appears to havegreater tissue specificity than previously thought, as they are able torecruit FH/FHL-1 through their CCP7 domains and not FH’s secondaryanchoring site found in CCPs19-20 (Clark et al., J Immunol (2013)190:2049-2057). This is likely to be an evolutionary twist, as it hasbeen discovered that the main regulator of complement within BrM is thetruncated FHL-1 protein (Clark et al J. Immunol (2014) 193, 4962-4970),which only has the one surface anchoring site in CCP7 and lacksCCPs19-20. In contrast, the Y402H polymorphism is not associated withkidney disease where the CCP19-20 domain of FH is known to be the mainGAG-mediated anchoring site (Clark et al., J Immunol (2013)190:2049-2057). Age-related changes in the BrM expression levels of HSand DS, themselves considered part of the normal ageing process, havealso been associated with AMD, and may go some way as to explain theage-related nature of the genetically driven AMD.

A rare mutation (R1210C) in the C-terminal CCP19-20 region of FH, whichdoes not bind to BrM, has a very high level of association with AMD, andFH protein carrying this mutation is found covalently bound to albumin(Sanchez-Corral et al., Am J Hum Genet (2002) 71:1285-1295) preventingthe FH protein from leaving the circulation and entering eye tissue.Some research suggests that the large confluent drusen that precedegeographic atrophy and the associated pigmentary changes in the RPEindicate that dry AMD results firstly from dysfunction of the RPE withsecondary effects within the choroid (Bhutto and Lutty Mol Aspects Med(2012) 33:295-317). In contrast, Whitmore et al. reported changes in thechoriocapillaris preceding all forms of late-stage AMD including thedeposition of the terminal complement membrane attack complex (MAC), andargue that excessive complement activation in the choriocapillaris isthe primary event with RPE atrophy being secondary (Whitmore et al.,Prog Retin Eye Res (2015) 45:1-29). These data imply that a geneticpredisposition conferred by alterations in complement genes is tolerateduntil changes in both BrM and the underlying choriocapillaris come tothe fore. Whether these changes are age-related, driven by oxidativestress or a result of RPE cell dysfunction remains to be seen, butnaturally occurring changes in these structures are known to beage-related.

C3 and C3b

Complement component 3 (C3) is an immune system protein having a centralrole in innate immunity and the complement system. Processing of C3 isdescribed, for example, in Foley et al. J Thromb Haemostasis (2015) 13:610-618, which is hereby incorporated by reference in its entirety.Human C3 (UniProt: P01024; SEQ ID NO:18) comprises a 1,663 amino acidsequence (including an N-terminal, 22 amino acid signal peptide). Aminoacids 23 to 667 encode C3 β chain (SEQ ID NO:19), and amino acids 749 to1,663 encode C3 α′ chain (SEQ ID NO:20). C3 β chain and C3 α′ chainassociate through interchain disulphide bonds (formed between cysteine559 of C3 β chain, and cysteine 816 of the C3 α′ chain) to form C3b. C3ais a 77 amino acid fragment corresponding to amino acid positions 672 to748 of C3 (SEQ ID NO:21), generated by proteolytic cleavage of C3following activation through the classical complement pathway and thelectin pathways.

C3b is a potent opsonin, targeting pathogens, antibody-antigen immunecomplexes and apoptotic cells for phagocytosis by phagocytes and NKcells. C3b is also involved in the formation of convertase enzymecomplexes for activating and amplifying complement responses. C3bassociates with Factor B to form the C3bBb-type C3 convertase(alternative complement pathway), and can associate with C4b and C2a toform the C4b2a3b-type C5 convertase (classical pathway), or with C3bBbto form the C3bBb3b-type C5 convertase (alternative pathway).

Processing of C3b to the form iC3b, which is proteolytically inactiveand which cannot itself promote further complement amplification,involves proteolytic cleavage of the C3b α′ chain at amino acidpositions 1303 and 1320 to form an α′ chain fragment 1 (corresponding toamino acid positions 672 to 748 of C3; SEQ ID NO:22), an α′ chainfragment 2 (corresponding to amino acid positions 1321 to 1,663 of C3;SEQ ID NO:23). Thus, iC3b comprises the C3 β chain, C3 α′ chain fragment1 and C3 α′ chain fragment 2 (associated via disulphide bonds). Cleavageof the α′ chain also liberates C3f, which corresponds to amino acidpositions 1304 to 1320 of C3 (SEQ ID NO:24).

As used herein “C3” refers to C3 from any species and include isoforms,fragments, variants or homologues of C3 from any species. In someembodiments, the C3 is mammalian C3 (e.g. cynomolgous, human and/orrodent (e.g. rat and/or murine) C3). Isoforms, fragments, variants orhomologues of C3 may optionally be characterised as having at least 70%,preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity to the amino acid sequence ofimmature or mature C3 from a given species, e.g. human C3 (SEQ IDNO:18).

As used herein “C3b” refers to and includes isoforms, fragments,variants or homologues of C3b from any species. In some embodiments, theC3b is mammalian C3b (e.g. cynomolgous, human and/or rodent (e.g. ratand/or murine) C3b).

Isoforms, fragments, variants or homologues of C3b may optionally becharacterised as comprising a C3 α′ chain fragment 1, C3 α′ chainfragment 2 and a C3 β having at least 70%, preferably one of 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity to the amino acid sequences of the respectivepolypeptides from a given species, e.g. human. That is, the C3b maycomprise: a C3 α′ chain fragment 1 having at least 70%, preferably oneof 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity to SEQ ID NO:22; a C3 α′ chain fragment 2having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQID NO:23; and a C3 β chain having at least 70%, preferably one of 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity to SEQ ID NO:19.

Isoforms, fragments, variants or homologues of C3b may optionally befunctional isoforms, fragments, variants or homologues, e.g. having afunctional property/activity of the reference C3b, as determined byanalysis by a suitable assay for the functional property/activity. Forexample, Isoforms, fragments, variants or homologues of C3b may becharacterised by the ability to act as an opsonin, and/or to formfunctional C3/C5 convertase.

Complement Factor I

Processing of C3b to iC3b is performed by Complement Factor I (encodedin humans by the gene CFI). Human Complement Factor I (UniProt: P05156;SEQ ID NO:25) has a 583 amino acid sequence (including an N-terminal, 18amino acid signal peptide). The precursor polypeptide is cleaved byfurin to yield the mature Complement Factor I, comprising a heavy chain(amino acids 19 to 335), and light chain (amino acids 340 to 583) linkedby interchain disulphide bonds. Amino acids 340 to 574 of the lightchain encode the proteolytic domain of Complement Factor I (SEQ IDNO:26), which is a serine protease containing the catalytic triadresponsible for cleaving C3b to produce iC3b (Ekdahl et al., J Immunol(1990) 144 (11): 4269-74).

As used herein “Complement Factor I (FI)” refers to Complement Factor Ifrom any species and includes isoforms, fragments, variants orhomologues of Complement Factor I from any species. In some embodiments,the Complement Factor I is mammalian Complement Factor I (e.g.cynomolgous, human and/or rodent (e.g. rat and/or murine) ComplementFactor I).

Isoforms, fragments, variants or homologues of Complement Factor I mayoptionally be characterised as having at least 70%, preferably one of80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% aminoacid sequence identity to the amino acid sequence of immature or matureComplement Factor I from a given species, e.g. human Complement Factor I(SEQ ID NO:25). Isoforms, fragments, variants or homologues ofComplement Factor I may optionally be functional isoforms, fragments,variants or homologues, e.g. having a functional property/activity ofthe reference Complement Factor I (e.g. full-length human ComplementFactor I), as determined by analysis by a suitable assay for thefunctional property/activity. For example, an isoform, fragment, variantor homologue of Complement Factor I may display serine protease activityand/or may be capable of inactivating C3b.

Proteolytic cleavage of C3b by Complement Factor I to yield iC3b isfacilitated by co-factors for Complement Factor I. Co-factors forComplement Factor I typically bind to C3b and/or Complement Factor I,and potentiate processing of C3b to iC3b by Complement Factor I.

Complement Receptor 1

Complement Receptor 1 (CR1) acts as a cofactor for Complement Factor I,enabling cleavage of C3b to iC3b and downstream products.

iC3b does not amplify or activate the complement system, but it canstill act as an opsonin to target pathogens for phagocytosis. iC3bproduction therefore results in local immune system activation andinflammatory effects. This can have negative consequences for sufferersof complement-related disease and may contribute to the development orworsening of an existing complement-related disease/condition.

Factor H (FH) and truncated FH isoform FHL-1 act as co-factors for FI toproduce iC3b, but they cannot promote further degradation of iC3b whichcan lead to undesirable iC3b accumulation. In addition, the accumulationof iC3b may contribute to further debris in the affected area, leadingto e.g. the (further) development of drusen in macular degeneration.

In contrast, CR1 and the polypeptides according to the presentinvention, see e.g. FIGS. 2, 3B, 7A, can act in combination with FI topromote further breakdown of iC3b into advantageous downstream productssuch as C3c, C3dg and C3b. These molecules are not opsonins and thusavoid recruiting immune system components. Their presence in an affectedarea is preferable to iC3b accumulation.

As used herein, “Complement Receptor 1 (CR1)” refers to CR1 from anyspecies and includes isoforms, fragments, variants or homologues of CR1from any species. In some embodiments, the CR1 is mammalian CR1 (e.g.cynomolgous, human and/or rodent (e.g. rat and/or murine) CR1).

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

The features disclosed in the foregoing description, or in the followingclaims, or in the accompanying drawings, expressed in their specificforms or in terms of a means for performing the disclosed function, or amethod or process for obtaining the disclosed results, as appropriate,may, separately, or in any combination of such features, be utilised forrealising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplaryembodiments described above, many equivalent modifications andvariations will be apparent to those skilled in the art when given thisdisclosure. Accordingly, the exemplary embodiments of the invention setforth above are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations providedherein are provided for the purposes of improving the understanding of areader. The inventors do not wish to be bound by any of thesetheoretical explanations.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

Where a nucleic acid sequence in disclosed the reverse complementthereof is also expressly contemplated.

The following numbered paragraphs (paras) describe particular aspectsand embodiments of the present invention:

-   1. A polypeptide having at least 80% sequence identity to SEQ ID    NO:4, wherein the polypeptide has a length of 700 amino acids or    fewer.-   2. The polypeptide according to para 1, wherein the polypeptide has    a length of 50 to 700 amino acids.-   3. The polypeptide according to para 1 or 2, wherein X₁ is A or T,    X₂ is P or L, and/or X₃ is G or R.-   4. The polypeptide according to any one of paras 1 to 3, comprising    SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:13.-   5. The polypeptide according to any one of paras 1 to 4, consisting    of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:13.-   6. The polypeptide according to any one of paras 1 to 5, which is    capable of binding to C3b.-   7. The polypeptide according to any one of paras 1 to 6, which binds    to C3b in the region bound by a co-factor for Complement Factor I.-   8. The polypeptide according to any one of paras 1 to 7, which binds    to C3b in the region bound by Complement Receptor 1 (CR1).-   9. The polypeptide according to any one of paras 1 to 8, which acts    as a co-factor for Complement Factor I.-   10. The polypeptide according to any one of paras 1 to 9, which is    capable of diffusing across Bruch’s membrane (BrM).-   11. The polypeptide according to any one of paras 1 to 10, which is    not glycosylated or is partially glycosylated.-   12. The polypeptide according to any one of paras 1 to 11, wherein    the amino acid sequence comprises one or more amino acid    substitutions at position 509, 578, 959 and/or 1028 (numbered    according to Uniprot: P17927).-   13. The polypeptide according to para 12, wherein the one or more    amino acid substitutions are selected from N509Q, N578Q, N959Q    and/or N1028Q (numbered according to Uniprot: P17927).-   14. The polypeptide according to any one of paras 1 to 13,    comprising, or consisting, of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ    ID NO:15.-   15. The polypeptide according to any one of paras 1 to 14,    additionally comprising a secretory pathway sequence.-   16. The polypeptide according to para 15, wherein the secretory    pathway sequence comprises SEQ ID NO:7.-   17. The polypeptide according to para 15 or para 16, wherein the    polypeptide additionally comprises a cleavage site for removing the    secretory pathway sequence.-   18. A nucleic acid encoding the polypeptide according to any one of    paras 1 to 17.-   19. A vector comprising the nucleic acid of para 18.-   20. A cell comprising the polypeptide according to any one of paras    1 to 17, the nucleic acid according to para 18, or the vector    according to para 19.-   21. A method for producing a polypeptide, comprising introducing    into a cell a nucleic acid according to para 18 or a vector    according to para 19, and culturing the cell under conditions    suitable for expression of the polypeptide.-   22. A cell which is obtained or obtainable by the method according    to para 21.-   23. A pharmaceutical composition comprising the polypeptide    according to any one of paras 1 to 17, the nucleic acid according to    para 18, the vector according to para 19, or the cell according to    para 20 or 22, optionally comprising a pharmaceutically acceptable    carrier, adjuvant, excipient, or diluent.-   24. The polypeptide according to any one of paras 1 to 17, the    nucleic acid according to para 18, the vector according to para 19,    the cell according to para 20 or 22, or the pharmaceutical    composition according to para 23, for use in a method of treating or    preventing a disease or condition.-   25. Use of the polypeptide according to any one of paras 1 to 17,    the nucleic acid according to para 18, the vector according to para    19, the cell according to para 20 or 22, or the pharmaceutical    composition according to para 23, in the manufacture of a medicament    for treating or preventing a disease or condition.-   26. A method of treating or preventing a disease or condition,    comprising administering to a subject the polypeptide according to    any one of paras 1 to 17, the nucleic acid according to para 18, the    vector according to para 19, the cell according to para 20 or 22, or    the pharmaceutical composition according to para 23.-   27. A method of treating or preventing a disease or condition in a    subject, comprising modifying at least one cell of the subject to    express or comprise a polypeptide according to any one of paras 1 to    17, a nucleic acid according to para 18, or a vector according to    para 19.-   28. The polypeptide, nucleic acid, vector, cell, or pharmaceutical    composition for use according to para 24, the use according to para    25, or the method according to para 26 or para 27, wherein the    disease or condition is a disease or condition in which C3b or a    C3b-containing complex, an activity/response associated with C3b or    a C3b-containing complex, or a product of an activity/response    associated with C3b or a C3b-containing complex is pathologically    implicated.-   29. The polypeptide, nucleic acid, vector, cell, or pharmaceutical    composition for use, the use, or the method according to any one of    paras 24 to 28, wherein the disease or condition is age-related    macular degeneration (AMD).-   30. The polypeptide, nucleic acid, vector, cell, or pharmaceutical    composition for use, the use, or the method according to any one of    paras 24 to 29, wherein the method for treating or preventing a    disease or condition comprises modifying at least one retinal    pigment epithelial (RPE) cell of the subject to express or comprise    a polypeptide according to any one of paras 1 to 17, a nucleic acid    according to para 18, or a vector according to para 19.-   31. A kit of parts comprising a predetermined quantity of the    polypeptide according to any one of paras 1 to 17, the nucleic acid    according to para 18, the vector according to para 19, the cell    according to para 20 or para 22 or the pharmaceutical composition    according to para 23.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures.

FIGS. 1A and 1B. CR1a and nCR1a protein expression from human HEK293cells. (1A) CR1a is expressed as two glyco-forms that are reduced to onelower MW band after deglycosylation treatment with PNGase. (1B) nCR1a isexpressed as single form of same MW as deglycosylated CR1a.

FIG. 2 . The ability of CR1a and nCR1a expressed and secreted by humancells to act as cofactors for Factor I-mediated breakdown of C3b.CR1a/nCR1a + FI + C3b reactions produced iC3b and C3dg (lanes 7 and 11).

FIGS. 3A to 3C. (3A) Presence of CR1a in the diffusion chamber of anUssing chamber after 24 hours, showing ability to diffuse throughBruch’s membrane (BrM). (3B, 3C) The ability of (3B) CR1a and (3C) nCR1ato act as cofactors for Factor I to break down C3b after polypeptidediffusion through Bruch’s membrane (BrM).

FIG. 4 . Schematic of an Ussing chamber used in diffusion experiments,in which (a) is enriched Bruch’s membrane from human donor eyes,covering a 5 mm aperture and representing the only passage of liquidfrom one chamber to another; (b) are sampling access points; and (c) aremagnetic stirrer bars.

FIG. 5 . Binding kinetics of CR1a, FH and FHL-1 for C3b measured byBiolayer interferometry (BLI).

FIG. 6 . Detection of secreted CR1a (AAV-CR1a media) from the tissueculture media of AAV-CR1a transduced ARPE-19 cells using anti-CR1aantibody. Recombinant CR1a protein was used as a positive control. Mediaalone and media from culture of AAV-GFP transduced ARPE-19 cells wereused as negative controls.

FIGS. 7A and 7B. Ability of secreted CR1a from human ARPE-19 cells toact as a cofactor for Factor I-mediated breakdown of C3b. (7A) SecretedCR1a acts as FI cofactor to produce iC3b (product e) and C3dg (productf). CR1a secreted from human HEK293 cells was provided as a control.(7B) Cultured AAV-CR1a transduced RPE cells showed increased ability tobreak down C3b (product a) to iC3b (product b) compared to mediacontaining non-transduced cells.

FIG. 8 . Schematic of the macular region of the eye, showingphotoreceptors, retinal pigment epithelium, Bruch’s membrane, and thechoriocapillaris and intercapillary septa in the choroid.

FIG. 9 . Representative fluorescence microscopy image of retinal tissuefrom Forest et al. (2015) Dis. Mod. Mech. 8, 421-427 (FIG. 1 ). Shownare drusen deposits underlying degraded RPE cells and areas ofcomplement activation. Scale bar 20 µm.

FIG. 10 . Schematic of the macular region of the eye showing key stagesof localised expression of effective complement therapeutic.

FIG. 11 . Example of an expression vector comprising nucleic acidencoding a polypeptide according to the present invention (e.g. CR1a),promoter elements, replication elements and selection elements.

EXAMPLES

In the following Example(s), the inventors describe the design ofrecombinant CR1 proteins comprising the C3b binding co-factor regions ofComplement Receptor 1. Also described is the ability of these proteinsto be expressed by human cells, diffuse through Bruch’s membraneenriched from human donor eyes, and confer regulatory activity, i.e.facilitate the FI-mediated breakdown of C3b into iC3b and furtherbreakdown products.

Example 1

DNA inserts encoding the amino acid sequences shown in SEQ ID NOs:2 and5 were prepared by recombinant DNA techniques, and cloned into a vectorto generate constructs for recombinant expression of CR1 peptides. Theamino acid sequences and features thereof are shown below:

CR1a (SEQ ID NO:47)MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRI PCGLPPTIANGDF ISTNRENFHYGSVVTYRCNPGSGGRKVFEL VGEPSIYCTSN DDQVGIWSGPAPQCII

Signal peptide (SEQ ID NO:7); CCPs8-10 of CR1 (SEQ ID NO:2) (UniProt:P17927, residues 491-685)

nCR1a (SEQ ID NO:48)MRLLAKIICLMLWAICVAGHCQAPDHFLFAKLKTQTQASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRIQYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRI PCGLPPTIANGDF ISTNRENFHYGSVVTYRCNPGSGGRKVFEL VGEPSIYCTSN DDQVGIWSGPAPQCII

Signal peptide (SEQ ID NO:7); CCPs8-10 of CR1 (SEQ ID NO:5) (UniProt:P17927, residues 491-685) comprising substitutions N509Q and N578Q

The 18-amino acid signal peptide is designed to be cleaved from thepolypeptides upon secretion.

In some experiments, HIS-tagged CR1a and nCR1a were used, e.g. as shownin SEQ ID NO:40 and 21, respectively.

Expression of Protein From Human Cells

HEK 293T cells (7×10⁶ cell per plate) were grown in 15 cm culture platesovernight in 17ml of Dulbecco’s Modified Eagle’s Medium with highglucose (DMEM, Sigma, catalogue number D469) supplemented with 10%Foetal Bovine Serum (FBS, Sigma, catalogue number F9665) in 5% CO₂incubator at 37° C. Once the cells reached 60% confluence they weretransiently transfected with 14.4 µg plasmid expressing either CR1a (SEQID NO:2) or nCR1a (SEQ ID NO:5) linked to the signal peptide (SEQ IDNO:7) with 86.4µI of 7.5 mM Polyethylenimine (PEl, Polysciences,catalogue number 24765-2) 150 mM NaCl (Fisher Scientific UK Ltd,catalogue number 1073592). For the negative control 14.4 µI of Tris-EDTAbuffer was used instead of the plasmid DNA. Five hours aftertransfection, the transfection medium containing 10% FBS was replacedwith 17.5 ml fresh DMEM with high glucose supplemented with 2% FBS(referred to henceforth as expression media). Expression media wascollected 24, 48, 72 and 140 hours after transfection. 80 µI of 0.5 MPhenylmethanesulfonyl fluoride (PMSF, Sigma, Catalogue number P7626) wasadded to every 100 ml of expression media collected and stored at 4° C.Expression media collected after 24 hrs was used for diffusion andfunction studies described below.

Characterisation of Secreted Protein From Human Cells

Protein expression from human cells is shown in FIGS. 1A and 1B.Recombinantly expressed and purified CR1a protein was found to besecreted from the cells as two glyco-forms. Treatment with PNGaseF, anenzyme that removes glycosylation, reduced the two bands to one band oflower apparent molecular weight (1A). A non-glycosylated form of theprotein (nCR1a) was expressed. Western blotting demonstrated that nCR1aproduces a single band that migrates to the same position as theenzymatically de-glycosylated protein (1B).

C3b Breakdown Activity

CR1a and nCR1a expressed and secreted from human HEK293 cells weretested for their ability to act as cofactors for Factor I-mediatedbreakdown of C3b.

1 µg of recombinant CR1a or nCR1a protein was mixed with 2 µg of pureC3b protein and 0.04 µg of pure complement Factor I (FI; VWRInternational, catalogue no. 341280) for 15 minutes at 37° C. FHL-1 wasprovided as a co-factor control for FI. CR1a/nCR1a, CR1 a/nCR1a+FI,CR1a/nCR1a+C3b and C3b alone were also provided as controls. Reactionswere stopped with the addition of 4x SDS-loading buffer and heating to100° C. for 5 minutes. Samples were then run on 4-12% NuPAGE Bis-Trisgels, run at 200V for 60 minutes. Samples were transferred ontonitrocellulose membranes at 80mA for 1.5 hours using semi-dry transferapparatus in transfer buffer (25 mM Tris, 192 mM glycine, 10% (v/v)Methanol). The membranes were the blocked in PBS, 10% (w/v) milk, 0.2%(w/v) BSA for 16 hours at 4° C. before the addition of anti-C3b antibody(HycultBiotech, catalogue no. HM2287), at 100 µg/ml, in PBS, 0.2% (v/v)Tween-20 (PBS-T) for 1 hours at room temperature. Membranes were washed2x 30 min in PBS-T before the addition of a 1:2500 dilution of HRPconjugated goat antimouse for 1 hour at room temperature, protected fromlight. Membranes were washed 2x 30 min in PBS-T before the addition ofSuperSignal West Pico Chemiluminescent Substrate (Thermo FisherScientific, catalogue no. 34080) for 3 min at room temperature. Reactivebands were detected by exposing Super RX-N X-ray film (FujiFilm,catalogue no. PPB5080) to the treated membrane for 2 min at roomtemperature, and developed on an automated X-ray film developer.

The results are shown in FIG. 2 . Both secreted proteins were found tobe able to act as cofactors for Factor I, leading to the breakdown ofC3b to firstly iC3b (a₁), and further to C3dg (a₁₋₁). The C3b breakdownusing CR1a/nCR1a continues further than the normal native breakdown ofC3b observed using FI + FHL-1 (second lane), which only produces iC3b(a₁).

Ussing Chamber Diffusion Experiments and C3b Breakdown Activity

The macular region of enriched Bruch’s membrane isolated from donor eyeswas mounted in an Ussing chamber (Harvard Apparatus, Hamden, CT) asdescribed in McHarg et al., J Vis Exp (2015) 1-7, supra. Once mounted,the 5-mm-diameter macular area was the only barrier between twoidentical compartments, i.e. liquid must pass through Bruch’s membrane(FIG. 4 ). Both sides of Bruch’s membrane were washed with 2 ml PBS for5 min at room temperature. The structural integrity of Bruch’s membranewas tested prior to the experiments by its ability to retain 2 ml ofliquid in one chamber without leaking into the next. 2 ml of expressionmedia containing recombinant protein (CR1a and/or nCR1a, see above) wasadded to a chamber (henceforth designated the sample chamber) and 2 mlof fresh PBS was added to the other chamber (henceforth referred to asthe diffusate/diffusion chamber). The Ussing chamber was left at roomtemperature for 24 hours with gentle stirring in each compartment withmagnetic stirrer bars to avoid generating gradients of diffusingprotein.

CR1a was added to the sample chamber. After 24 hours, samples from eachchamber (original sample chamber and diffusion chamber) were analysedfor the presence of CR1a.

The results are shown in FIG. 3A. CR1a was found to be present in thediffusion chamber after 24 hours.

Separately, CR1a and nCR1a were added to sample chambers and samplesfrom both chambers were analysed for C3b-breakdown activity. After 24hours, 18.6µI samples were taken from each chamber and mixed with 1µI (1µg) of pure C3b protein and 0.4µI of pure complement factor 1 (0.04 µg,VWR International, catalogue no. 341280) for one of 15, 30, or 60minutes at 37° C. Reactions were stopped with the addition of 4xSDS-loading buffer and heating to 100° C. for 5 minutes. Samples werethen run on 4-12% NuPAGE Bis-Tris gels, run at 200V for 60 minutes.

The results are shown in FIGS. 3B and 3C. Breakdown of C3b in thediffusion chamber demonstrates that both glycosylated CR1a (3B) andnon-glycosylated nCR1a (3C) were able to cross Bruch’s membrane from thesample chamber and remain functionally active. CR1a and nCR1a from bothchambers were found to act successfully as co-factors for FI to breakdown C3b into proteolytically-inactive C3b (iC3b) and further products,as demonstrated by the presence of bands representing C3b breakdownproducts iC3b and C3dg. Thus, CR1a and nCR1a are able to diffuse throughBrM and retain the ability to confer C3b breakdown in the presence ofFactor I.

Example 2

The expression levels of polypeptides according to the present inventionare compared to assess if there is an optimal formulation, i.e. whetherglycosylated polypeptides express at higher levels than non-glycosylatedpolypeptides. It is anticipated that the glycosylation state of thepolypeptides will have a minimal effect on level of expression.

CR1a was found to be expressed well by human cells. nCR1a was found tobe expressed at lower levels than CR1a but both polypeptides were foundto be functionally active, see e.g. FIG. 2 .

Example 3

The binding kinetics of purified polypeptides according to the presentinvention for C3b is tested using Surface Plasmon Resonance (SPR),whereby C3b is immobilised onto SPR chips and the polypeptides used inthe fluid phase. The association and dissociation constants are directlymeasured and a kD value for the interaction inferred.

Whilst no differences are observed in the binding of the CR1a (CCPs8-10) or CR1b (CCPs 15-17) species to C3b, glycosylated polypeptidesbind more strongly to C3b than non-glycosylated polypeptides.

Binding Kinetics Measured by Bio-layer Interferometry (BLI)

The affinity of CR1a for C3b protein was measured using OctetRed96System (ForteBio, Pall Corp., USA). Biotinylated C3b protein was dilutedin 0.2% PBST to final concentration of 0.4 µg/mL and loaded onto HighPrecision Streptavidin (SAX) Biosensors (ForteBio, Pall Corp., USA) for600 s, previously hydrated with the same buffer for 20 min. C3b-loadedsensors were then washed with 0.2% PBST for 150 s (baseline) and dippedinto wells containing CR1a in different concentrations ranging from 30.0µg/mL to 2.6 µg/mL for 600 s (association) followed by wash with 0.2%PBST for 600 s (dissociation). Association and dissociation profileswere recorded and analysed with ForeBio Data Analysis v9 (ForteBio, PallCorp., USA). The negative control i.e. 0.2% PBST containing well wasused in parallel to subtract binding resulting from nonspecificinteractions with the sensors. Experiments were performed using thekinetics mode, at 25° C. and sample plates were pre-agitated for 3 min.The binding profiles were globally fitted to 1:1 (one analyte insolution to one binding site on the surface). The KD was determinedusing data of the association (from 0 s to 600 s) and dissociation (from0 s to 100 s) phases from four the lowest available analyteconcentration by steady-state analysis. Binding affinities were alsodetermined for native soluble C3b-binding complement regulators Factor H(FH) and FHL-1 to immobilised C3b.

The results are shown in FIG. 5 . The binding affinity of CR1a for C3bwas found to be 21 nM. For comparison, the binding affinity for C3b ofFH was 580 nM and of FHL-1 was 1.2 mM. CR1a therefore bindssignificantly more strongly to C3b than either native soluble complementregulator. The strong binding affinity of CR1a for C3b means that CR1ais a more effective agent to promote C3b break down than agents based onFH or FHL-1.

A strong binding affinity also enables CR1a to promote degradation ofiC3b into desirable further downstream products e.g. C3dg (see FIG. 2 ).In contrast, FH and FHL-1 cannot cause degradation of C3b beyond iC3b.iC3b is a pro-inflammatory molecule which acts to recruit immune cellsto the site of complement activation, which in turn cause negativeinflammatory effects. Further breakdown of iC3b to C3dg by CR1a is thusadvantageous and avoids further damage caused by the immune system.

Example 4

The diffusion rates of polypeptides according to the present inventionare compared using Ussing chamber experiments (described above) to seeif any differences arise due to the formulation of the protein.

Experiments include using Bruch’s membranes derived from donors with AMDto determine whether the material deposited in the Bruch’s membrane inthis condition, including drusen and basal linear deposits, compromisesthe ability of the polypeptide to cross Bruch’s membrane. It isanticipated that the optimal polypeptide will cross Bruch’s membraneefficiently even in the presence of AMD changes.

Example 5

Nucleic acid according to the present invention, optionally encoding thepreferred signal peptide, a polypeptide having at least 80% sequenceidentity to SEQ ID NO:4 and a termination codon, is inserted into an AAVvector. The resultant expression vector is used to transfect culturedRPE cells. The expression and secretion of the encoded polypeptide isevaluated. It is anticipated that the polypeptide having at least 80%sequence identity to SEQ ID NO:4 is secreted by the RPE cells and thatthe signal peptide has been cleaved from the secreted polypeptide.

Adeno-associated virus (AAV) transductionNucleic acid encoding the CR1apolypeptide described in Example 1 was transfected into human APRE-19cells (ATCC, USA) derived from retinal pigment epithelium.

AAV2 serotype viral particles were pre-packed with CR1a plasmid. ARPE-19cells were seeded on six well cell culture plate (Corning) in 2 ml ofDMEM/F12 growth medium (ATCC, USA) supplemented with 10% (v/v) fetalbovine serum (ATCC, USA) at the density of 300,000 cells per well. Cellswere then left for incubation at 37° C. in humidified atmosphere of 5%CO₂ for 24 hrs. After incubation, cells were washed twice with 2 mlserum free DMEM/F12 growth medium. AAV2-CR1a at multiplicity ofinfection (MOI) 100,000 in serum free DMEM/F12 growth medium in totalvolume of 1 ml was added.

AAV2-CR1a containing medium was incubated with cells for 24 hrs (37° C.,5% CO₂) followed by replacement with 2 ml of fresh serum free DMEM/F12growth medium the next day. Control cells transduced with AAV-GFP weregrown in parallel. Transduction efficiency was assessed 14 dayspost-infection. Secretion of CR1a by ARPE-19 cells was detected by dotblot: conditioned media from CR1a-transduced RPE cells was contactedwith in-house polyclonal anti-CR1a antibody.

The results are shown in FIG. 6 . Immunoreactivity with the anti-CR1aantibody was seen in both samples tested (AAV-CR1a media), compared tomedia from RPE cells transduced with AAV-GFP (Ctrl media) used as anegative control. Purified recombinant CR1a protein was included as apositive control.

Recombinant CR1a polypeptides secreted from human APRE-19 and HEK293cells were assessed for their ability to break down C3b to iC3b andC3dg.

The results are shown in FIG. 7A. Functional CR1a polypeptide secretedfrom human APRE-19 cells was found to act as a cofactor for Factor Ileading to breakdown of C3b into iC3b (product e) and C3dg (product f).Reaction containing FHL-1+FI+C3b provided a MW control for C3b andproduct iC3b.

C3b break down was assessed in tissue culture media of human RPE cells(ARPE-19) transduced with AAV-delivered CR1a. AAV-GFP transduced RPEcells were used as a negative control. Transduced cells weresupplemented with purified C3b and Factor I 14 days after transduction.C3b and iC3b levels in tissue culture media were detected by Westernblot.

The results are shown in FIG. 7B. RPE cells produce their own complementcomponents and were found to have a low native turnover of C3b (producta) into iC3b (product b; lane 2). However, RPE cells expressingAAV-delivered CR1a were found to have increased C3b breakdown capacityto iC3b (product b; lane 3) compared to the native turnover rate.

This indicates that CR1a is secreted successfully from eye cells, e.g.retinal pigment epithelium cells, is functionally active as a FIcofactor, enhances C3b break down to downstream products, is capable ofcomplement regulation, and would provide therapeutic benefit forconditions involving over-activation of complement e.g. an excess ofC3b.

Example 6

The polypeptides of the present invention find use in methods oftreatment or prevention of complement-related disorders. One example ofa complement related disorder is macular degeneration in the eye, e.g.AMD.

FIG. 8 provides a schematic of the eye macular region. The retinalpigment epithelium (RPE) is a continuous monolayer of cuboidal/columnarepithelial cells between the neurosensory retina and the vascularchoroid. The cells have physical, optical, metabolic/biochemical andtransport functions and play a critical role in the normal visualprocess. The RPE is separated from the choroid by Bruch’s membrane(BrM)— a thin (2-4 µm), acellular, five-layered, extracellular matrix.The BrM serves two major functions: the substratum of the RPE and ablood vessel wall. Immediately adjacent to the BrM, and within thechoroid, is a layer of capillaries termed the choriocapillaris.Complement activation centres on the extracellular matrix of thechoriocapillaris, termed the intercapillary septa.

The hallmark lesions of AMD, drusen, are formed from the accumulation oflipids and cellular debris, including many complement activationproducts. Drusen develop within the BrM adjacent to the RPE layer anddisrupt the flow of nutrients from the choroid to the RPE, leading tocell dysfunction and death. The death of RPE cells also causesdysfunction of photoreceptor cells and subsequent loss of visual acuity.

A representative fluorescence microscopy image of drusen deposits isprovided in FIG. 9 , taken from Forest et al. (2015) Dis. Mod. Mech. 8,421-427 (FIG. 1 ). FIG. 9 shows retinal tissue from an 82-year-oldfemale with AMD. A cell membrane marker shows degraded RPE cellsoverlying drusen. Areas of complement activation within the drusen andaround the blood vessels are indicated by the terminal complementcomplex marker C5b-9. Nuclei are stained. Scale bar 20 µm.

Systemic administration of a complement-regulating molecule wouldrequire a high dose and carries a substantial risk of detrimentaloff-target effects on functioning complement systems. Localisedadministration, e.g. expression from RPE cells, is a safer and moreeffective delivery method. However, for an RPE-expressed molecule to beeffective in treating/preventing a complement-related disorder in theintercapillary septa of the choriocapillaris, i.e. the site ofcomplement over-activation, the molecule will have to cross Bruch’smembrane (see FIG. 10 ). That is, an effective complement-inhibitingtherapy must meet the requirements of all three stages in FIG. 10 .

As demonstrated herein, CR1a is:

-   1. Secreted from human RPE cells (see e.g. FIG. 6 );-   2. Able to passively diffuse across human BrM (see e.g. FIG. 3 );    and-   3. Able to mediate the breakdown of C3b into iC3b, and further    desirable break down products, in the presence of complement factor    I (see e.g. FIGS. 2, 3, 7 ).

Therefore, CR1a is able to be expressed by RPE cells, is able to reachthe areas of complement activation where C3b regulation is necessary,and can act as an effective therapeutic agent to treat complementover-activation in the eye, e.g. in AMD.

Example 7 Laser-Induced Choroidal Neovascularisation Model

This model applies laser burns to the retinas of mice or rats to inducechoroidal neovascularization, for example as described in Schnabolk etal, Mol Ther Methods Clin Dev. 2018; 9: 1-11. The size of the choroidalneovascular complexes can be measured by fluorescein angiography or byhistology.

Rodents are given a subretinal injection of AAV vector, e.g. AAV2,containing CR1a cDNA, or empty AAV vector as a control. Rodentsreceiving AAV vector containing CR1a cDNA secrete CR1a protein fromtheir retinal pigment epithelial cells. When maximal CR1a proteinexcretion is achieved, laser burns are applied to the rodent retinas andthe size of the choroidal neovascular complexes is measured at apre-specified time after the laser burns. The complement inhibitoryeffect of CR1a polypeptide is found to decrease the size of thechoroidal neovascular complexes compared to rodents administered theempty AAV vector.

Sodium Iodate Induced Retinal Degeneration

Mice are injected intravenously with sodium iodate which induces adegeneration of the retinal pigment epithelium that is partiallydependent upon complement activation, for example as described inKatschke et al., Sci Rep. 2018; 8(1):7348. CR1a is delivered byintravitreal injection as a recombinant protein or by subretinaldelivery using AAV vector, e.g. AAV2, containing CR1a cDNA. Empty AAVvector is administered as a control. CR1a treated mice are found to haveless retinal degeneration than the control mice that did not receiveCR1a treatment.

1-48. (canceled)
 49. An isolated nucleic acid encoding a polypeptidewhich is capable of binding to C3b, wherein the nucleic acid comprises anucleotide sequence having at least 85% sequence identity to SEQ IDNO:35, and wherein the encoded polypeptide has a total length of 450amino acids or fewer.
 50. An isolated nucleic acid according to claim49, wherein the nucleic acid is codon-optimised for expression in amammalian cell.
 51. An isolated nucleic acid according to claim 50,wherein the mammalian cell is a cell of the eye, optionally a retinalpigment epithelial (RPE) cell.
 52. An isolated nucleic acid according toclaim 49, wherein the encoded polypeptide has a total length of 250amino acids or fewer.
 53. An isolated nucleic acid according to claim49, wherein the nucleic acid comprises a nucleotide sequence having atleast 95% sequence identity to SEQ ID NO:35.
 54. An isolated nucleicacid according to claim 49, wherein the encoded polypeptide or aminoacid sequence comprises or consists of SEQ ID NO:2 or SEQ ID NO:3. 55.An isolated nucleic acid according to claim 49, wherein the nucleotidesequence comprises a sequence encoding a secretory pathway sequence. 56.An isolated nucleic acid according to claim 55, wherein the amino acidsequence of the secretory pathway sequence comprises, or consists of,SEQ ID NO:7.
 57. An isolated nucleic acid according to claim 55, whereinthe encoded polypeptide or amino acid sequence comprises a cleavage sitefor removing the secretory pathway sequence.
 58. An isolated nucleicacid according to claim 55, wherein the encoded polypeptide or aminoacid sequence comprises, or consists of, SEQ ID NO:47 or
 49. 59. Anisolated nucleic acid according to claim 49, wherein the nucleic acidcomprises nucleotides 67-648 of SEQ ID NO:35.
 60. An isolated nucleicacid according to claim 49, wherein the nucleic acid comprisesnucleotides 13-648 of SEQ ID NO:35.
 61. An isolated nucleic acidaccording to claim 49, wherein the nucleic acid comprises nucleotides7-651 of SEQ ID NO:35.
 62. A vector comprising the isolated nucleic acidaccording to claim
 49. 63. A vector according to claim 62, wherein thevector is a viral vector.
 64. A vector according to claim 62, whereinthe vector is a lentiviral vector, a retroviral vector, agammaretroviral vector, an adenoviral vector, an adeno-associated virus(AAV) vector, a vaccinia virus vector, or a herpesvirus vector.
 65. Avector according to claim 62, wherein the vector is an AAV vectorselected from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, orhybrids and/or mutants thereof.
 66. A vector according to claim 62,wherein the vector is an AAV2 vector, or a hybrid and/or mutant thereof.67. A mammalian cell comprising or expressing the nucleic acid accordingto claim
 49. 68. The mammalian cell according to claim 67, wherein themammalian cell is a cell of the eye, optionally a retinal pigmentepithelial (RPE) cell.